Supplementary Information for Single-cell sequencing of the small-rna transcriptome

Transcription

1 Supplementary Information for Single-cell sequencing of the small-rna transcriptome Omid R. Faridani 1,6,*, Ilgar Abdullayev 1,2,6, Michael Hagemann-Jensen 1,3, John P. Schell 4, Fredrik Lanner 4,5 and Rickard Sandberg 1,2,* 1 Ludwig Institute for Cancer Research, Stockholm, Sweden. 2 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden. 3 Respiratory Medicine Unit, Department of Medicine, Solna & Center for Molecular Medicine, Stockholm, Sweden. 4 Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden. 5 Division of Obstetrics and Gynecology, Karolinska University Hospital, Stockholm, Sweden. 6 These authors contributed equally Correspondence to: Omid.Faridani@licr.ki.se (O.R.F.) and Rickard.Sandberg@ki.se (R.S.) Contents Supplementary Figures Supplementary Tables Supplementary Software. 26

2 Supplementary Figure 1 Schematic outline of the single-cell small-rna library preparation. Single cells were lysed directly in the preparation tube and 5.8S rrna was masked for adapter ligation immediately by a complementary oligo. Then, the 3' adapter was ligated to the small RNAs and the free adapter was removed enzymatically prior to the 5' adapter ligation. The 5' adapter contains a unique molecular identifier (UMI) to remove PCR replicates. Sequences required for Illumina cluster generation and sample indexing were added through two rounds of PCR amplifications.

3 Supplementary Figure 2 Schematic illustration of the analysis pipeline for small-rna libraries.

4 Supplementary Figure 3 Estimation of the sequencing errors and evaluation of the UMI correction method. We designed seven synthetic RNA oligos that we subjected to library preparation and subsequently sequenced the resulting library. In the analyses, we treated their first eight nucleotides as UMIs and we calculated the percentage of mismatches that occurs during PCR amplifications and sequencing reaction. In the barplot we summarized the percentage of sequenced reads with mismatches before and after using the adjacency method to collapse reads to UMIs. The results show that the adjacency method correctly collapsed all single bp mismatches into molecules and partially corrected for the reads that harbored two or more mismatched within the UMIs. The error bars represent standard deviation. Adjacency procedure is available at

5 Supplementary Figure 4 Characteristics of small-rna libraries and length profiles of mrna-derived small-rnas detected in the single cells. (a) Numbers of genes per RNA species observed within the small RNAs libraries (18-40 nts). (b) Size distributions of mrna-derived small RNAs profiled in single cells and per cell type. Error bars denote standard deviations. Peaks in length distribution were observed around 18 and 31 nts suggesting that these RNAs were processed products and were not the result of mrna degradation.

6 Supplementary Figure 5 Comparison of mirna differential expression in the single-cell data and in previous bulk-rna analysis of naive and primed hescs. (a) Fraction of differentially expressed mirnas detected when comparing naive and primed hescs in bulk RNA measured by q-pcr (Sperber et al. Nat Cell Biol 2015) and in single-cell small-rna sequencing data (Fig 1h). (b) Scatter plot of mirnas expression in single naive and primed hescs (Fig 1h), colored according to their differential expression of bulk data in Sperber et al. Nat Cell Biol The mirnas identified as differentially expressed in single-cell data got colored according to whether the same mirna was identified as significantly higher expressed in Naive (green) or Primed (blue) hescs. Red circles denote mirnas that were found significantly differentially expressed in our study but not in Sperber et al.

7 Supplementary Figure 6 Scatter plot of small-rna expression in naïve and primed hescs. Analyses of tsrna, sdrna and mrna-derived small RNAs in naive and primed hescs. Genes in red were statistically significant, (assessed using SCDE: Single Cell Differential Expression analysis) requiring adjusted p-values below 0.05 and a 2-fold or larger difference in molecule counts between cell types. As in Fig 1h, 97 naïve and 90 primed cells were used.

8 Supplementary Figure 7 Variation of mirna expression in primed and naïve hescs. (a) Spearman coefficients are reported for the correlation of mirna expression among pairs of single cells within each cell type, as boxplots. Line in the middle of boxes represents median, and notches represent confidence interval around the median. Primed ESCs showed less correlation that was statistically significant among single cells compared to naive hescs (Welch Two Sample t-test). (b) mirna expression distributions across naive and primed hescs in absolute molecule counts, for the mirnas that showed biological variation in primed hescs (Fig 1i). These mirnas were abundant in naive hescs but were only detected at high levels in a few primed hescs. (c) Similar to (b) but for a selection of mirnas that were differentially expressed and also showed no significant variation within naive nor primed hescs. (a-c) Naive cells were sub-sampled according to the molecule distribution of primed cells in order to adjust for their differences in cell size.

9 Supplementary Figure 8 Variation of tsrna and sdrna expression in primed and naïve hescs. The absolute molecule counts data was normalized to adjust for the cell size difference, by sub-sampling molecules in primed cells to match the molecule distribution obtained for the smaller naive cells (see Materials and Methods for details). (a) Expression variation within primed and naïve hescs (as Fig 1i) for tsrnas and sdrnas. (b) Distribution of molecule counts observed in single naive and primed hescs, for the RNA genes with most variation within populations.

10 Supplementary Figure 9 Hierarchical clustering of single HEK293FT cells and human naive and primed ESCs. (a) Clustering of single cells (columns) based on expression of mirnas (rows). A number of mirnas (128) were expressed exclusively in naive hescs but not in HEK293FT nor primed hescs. (b) The mirnas were found uniquely expressed in naive hescs.

11 Supplementary Figure 10 Clustering of an extended number of cell types. Clustering of primed and naïve hescs, HEK293FT cells and glioblastoma cells using mirna expression with t-distributed stochastic neighbor embedding (t-sne). Analysis included total 398 cells and 585 mirnas expressed (>=1 molecule) in at least 2 cells. The number of cells used for each cell type is indicated inside parenthesis in the legend. Independent experimental replicates were performed for primed and naïve hescs in order to evaluate the batch effect.

12 Supplementary Figure 11 mirnas detection in dilutions of HEK293FT RNA and in single cells. Scatter plots of mirna expression on diluted HEK293FT RNA (1ug, 100ng, 10ng, 1ng, 0.1 ng, 0.01ng). Three replicates of 1ug profiles on the y-axis were plotted against three replicates of profiles generated from lower amounts of starting total RNA (x-axis). In the last row, profiles from 1ng HEK293FT total RNA was compared to the profiles obtained on three individual HEK29FT cells. Spearman coefficients are reported in top right corners.

13 Supplementary Figure 12 Comparison of small-rna profiling using single-cell method and the NEBNext method. Small-RNA sequencing libraries were generated from 1000 ng of HEK293FT RNA and we excluded the size selection step in the NEBNext protocol to make it directly comparable to our data (as we do not use any size selection step). Scatter plots summarized the profiling of mirnas (a), tsrnas (b) and sdrnas (c) using the NEBNext protocol (x-axis) against single-cell small-rna sequencing data (y-axis). Comparisons against different replicates generated with the same protocol are shown for NEBNext (d) and small-rna singlecell sequencing protocol (e). Spearman correlation was computed by adding pseudocount of 1 to all samples. Dropouts are seen as small RNAs only detected with one method. Genes with a 40-fold or larger difference in expression between methods were colored red (higher in single-cell small-rna protocol) and blue (higher in NEBNext protocol).

14 Supplementary Table 1: Lists significantly differentially expressed mirnas in naïve and primed hescs. Significant (adjusted p-value < 0.05 and abs(mle)>1) differentially expressed genes from SCDE. Maximum likelihood estimate (mle) represents the log(fold change of naïve vs primed hescs). Table is sorted by p-values (low to high). mirna name lb mle ub ce Z cz p- value adjusted p-value 101-3p -1,8753-1,5551-1,2807-1,2807-7,1608-7,0061 8,02E-13 2,45E p -3,6134-2,9273-2,3327-2,3327-7,1608-7,0061 8,02E-13 2,45E p -3,2932-2,8359-2,4699-2,4699-7,1608-7,0061 8,02E-13 2,45E a-3p -2,4699-2,1955-2,0125-2,0125-7,1608-7,0061 8,02E-13 2,45E b-3p -3,4305-3,0646-2,7444-2,7444-7,1608-7,0061 8,02E-13 2,45E b-5p -3,7964-3,1560-2,6986-2,6986-7,1608-7,0061 8,02E-13 2,45E a-5p -10,0170-8,4161-5,9461-5,9461-7,1608-7,0061 8,02E-13 2,45E p -8,3246-6,6322-4,1166-4,1166-7,1608-7,0061 8,02E-13 2,45E b-3p -2,2870-1,9211-1,6009-1,6009-7,1608-7,0061 8,02E-13 2,45E a-5p -1,5094-1,2807-1,0520-1,0520-7,1608-7,0061 8,02E-13 2,45E p -2,1040-1,6924-1,2807-1,2807-7,1608-7,0061 8,02E-13 2,45E p -3,1560-2,6986-2,3327-2,3327-7,1608-7,0061 8,02E-13 2,45E a-5p -3,4305-2,7444-2,1955-2,1955-7,1608-7,0061 8,02E-13 2,45E-12 18a-5p -2,6986-2,3785-2,1040-2,1040-7,1608-7,0061 8,02E-13 2,45E-12 18b-5p -11,0690-9,6053-6,6780-6,6780-7,1608-7,0061 8,02E-13 2,45E p -8,4161-3,5219-2,8816-2,8816-7,1608-7,0061 8,02E-13 2,45E b-5p -3,1560-2,3785-1,7838-1,7838-7,1608-7,0061 8,02E-13 2,45E b-3p -4,4367-3,8879-3,3390-3,3390-7,1608-7,0061 8,02E-13 2,45E-12 20b-5p -10,1999-8,7363-5,0314-5,0314-7,1608-7,0061 8,02E-13 2,45E p -4,5282-3,9336-3,5219-3,5219-7,1608-7,0061 8,02E-13 2,45E p -3,0646-2,3785-1,7838-1,7838-7,1608-7,0061 8,02E-13 2,45E p -7,3183-2,9273-2,1955-2,1955-7,1608-7,0061 8,02E-13 2,45E a-5p -3,0188-2,8359-2,6986-2,6986-7,1608-7,0061 8,02E-13 2,45E b-5p -1,9668-1,6466-1,3722-1,3722-7,1608-7,0061 8,02E-13 2,45E c-5p -3,8421-3,6134-3,4305-3,4305-7,1608-7,0061 8,02E-13 2,45E d-5p -2,2870-1,9211-1,6009-1,6009-7,1608-7,0061 8,02E-13 2,45E-12 30d-5p -2,4699-2,1498-1,9211-1,9211-7,1608-7,0061 8,02E-13 2,45E p -3,1560-2,6986-2,3327-2,3327-7,1608-7,0061 8,02E-13 2,45E p -2,5614-2,1955-1,8296-1,8296-7,1608-7,0061 8,02E-13 2,45E p -11,9838-8,2331-7,7300-7,7300-7,1608-7,0061 8,02E-13 2,45E p -8,0502-6,4035-3,8879-3,8879-7,1608-7,0061 8,02E-13 2,45E ,0251-3,4762-3,0188-3,0188-7,1608-7,0061 8,02E-13 2,45E p -2,8816-2,2412-1,6924-1,6924-7,1608-7,0061 8,02E-13 2,45E p -3,2018-2,2412-1,7838-1,7838-7,1608-7,0061 8,02E-13 2,45E-12

15 652-3p -2,0583-1,6009-1,2350-1,2350-7,1608-7,0061 8,02E-13 2,45E p -8,0502-6,3578-3,8421-3,8421-7,1608-7,0061 8,02E-13 2,45E p -8,4161-6,7237-4,2080-4,2080-7,1608-7,0061 8,02E-13 2,45E p -4,2080-3,6592-3,2018-3,2018-7,1608-7,0061 8,02E-13 2,45E p -2,4242-1,9211-1,5094-1,5094-7,1608-7,0061 8,02E-13 2,45E-12 92a-3p -1,9668-1,7381-1,5551-1,5551-7,1608-7,0061 8,02E-13 2,45E-12 92b-5p -3,6592-3,1560-2,7901-2,7901-7,1608-7,0061 8,02E-13 2,45E p -3,2475-2,9273-2,6072-2,6072-7,1608-7,0061 8,02E-13 2,45E ,2870-1,7381-1,2807-1,2807-7,1608-7,0061 8,02E-13 2,45E p 2,9273 3,6592 8,2789 2,9273 7,1608 7,0061 8,02E-13 2,45E p 3,9793 6,4950 8,6905 3,9793 7,1608 7,0061 8,02E-13 2,45E p 6,0376 6,4950 7,0439 6,0376 7,1608 7,0061 8,02E-13 2,45E ,6529 3,1103 3,5219 2,6529 7,1608 7,0061 8,02E-13 2,45E ,5614 2,8816 3,1560 2,5614 7,1608 7,0061 8,02E-13 2,45E p 4,4825 5,1686 9,5596 4,4825 7,1608 7,0061 8,02E-13 2,45E b-5p 4,3453 4,9399 5,5802 4,3453 7,1608 7,0061 8,02E-13 2,45E p 1,4637 1,7381 1,9668 1,4637 7,1608 7,0061 8,02E-13 2,45E p 2,0125 2,4242 2,8359 2,0125 7,1608 7,0061 8,02E-13 2,45E p 4,0251 4,8027 9,6053 4,0251 7,1608 7,0061 8,02E-13 2,45E p 2,8359 3,3847 8,8735 2,8359 7,1608 7,0061 8,02E-13 2,45E a-3p 3,1103 3,7964 8,4161 3,1103 7,1608 7,0061 8,02E-13 2,45E p 4,0251 6,4035 8,5990 4,0251 7,1608 7,0061 8,02E-13 2,45E p 4,6197 5,2601 7,5928 4,6197 7,1608 7,0061 8,02E-13 2,45E p 4,3453 5,0314 8,0959 4,3453 7,1608 7,0061 8,02E-13 2,45E a-3p 5,1228 5,7174 6,6322 5,1228 7,1608 7,0061 8,02E-13 2,45E a-5p 6,3578 7,2269 8,6905 6,3578 7,1608 7,0061 8,02E-13 2,45E p 6,1748 7,1354 8,3246 6,1748 7,1608 7,0061 8,02E-13 2,45E p 4,4825 5,1686 8,0044 4,4825 7,1608 7,0061 8,02E-13 2,45E p 5,5802 6,3121 7,2269 5,5802 7,1608 7,0061 8,02E-13 2,45E p 5,4430 6,0376 6,9524 5,4430 7,1608 7,0061 8,02E-13 2,45E a-3p 6,6322 7,3641 8,5076 6,6322 7,1608 7,0061 8,02E-13 2,45E b-3p 3,4762 4,2538 8,2789 3,4762 7,1608 7,0061 8,02E-13 2,45E c-3p 6,1748 6,9067 7,9587 6,1748 7,1608 7,0061 8,02E-13 2,45E a-3p 1,9211 2,2412 2,5157 1,9211 7,1608 7,0061 8,02E-13 2,45E p 3,0188 3,6592 8,9192 3,0188 7,1608 7,0061 8,02E-13 2,45E p 3,7506 6,4035 8,6448 3,7506 7,1608 7,0061 8,02E-13 2,45E p 6,9067 7, ,2914 6,9067 7,1608 7,0061 8,02E-13 2,45E p 3,8421 6,3578 8,5533 3,8421 7,1608 7,0061 8,02E-13 2,45E p 5,2143 5,8547 9,7425 5,2143 7,1608 7,0061 8,02E-13 2,45E p 5,2143 7,7757 9,9712 5,2143 7,1608 7,0061 8,02E-13 2,45E p 2,4699 3,2018 7,9587 2,4699 7,1608 7,0061 8,02E-13 2,45E p 3,8879 6,4035 8,5990 3,8879 7,1608 7,0061 8,02E-13 2,45E-12

16 487a-3p 2,6986 3,3390 8,6448 2,6986 7,1608 7,0061 8,02E-13 2,45E b-3p 4,5282 7,8215 9,7883 4,5282 7,1608 7,0061 8,02E-13 2,45E p 3,9793 4,5740 9,9255 3,9793 7,1608 7,0061 8,02E-13 2,45E p 3,0188 3,7964 8,6448 3,0188 7,1608 7,0061 8,02E-13 2,45E p 3,2018 3,8421 8,4161 3,2018 7,1608 7,0061 8,02E-13 2,45E ,2870 2,9731 3,8421 2,2870 7,1608 7,0061 8,02E-13 2,45E p 4,1623 4,4825 4,8027 4,1623 7,1608 7,0061 8,02E-13 2,45E p 2,7901 3,3847 3,9793 2,7901 7,1608 7,0061 8,02E-13 2,45E p 2,2412 2,8816 3,6592 2,2412 7,1608 7,0061 8,02E-13 2,45E a-5p 2,6986 2,9731 3,2018 2,6986 7,1608 7,0061 8,02E-13 2,45E b-5p 3,1560 3,4305 3,6592 3,1560 7,1608 7,0061 8,02E-13 2,45E p 3,3390 3,7964 4,2995 3,3390 7,1608 7,0061 8,02E-13 2,45E a-3p 2,7901 3,1103 3,4762 2,7901 7,1608 7,0061 8,02E-13 2,45E b-3p 2,7901 3,1103 3,4762 2,7901 7,1608 7,0061 8,02E-13 2,45E c-3p 2,7901 3,4305 4,2538 2,7901 7,1608 7,0061 8,02E-13 2,45E a-5p 3,0188 3,6134 9,1022 3,0188 7,1608 7,0061 8,02E-13 2,45E b 2,1955 2,6986 3,2475 2,1955 7,1608 7,0061 8,02E-13 2,45E c-5p 2,5614 3,1560 4,0251 2,5614 7,1608 7,0061 8,02E-13 2,45E d-5p 2,8816 3,2932 3,7964 2,8816 7,1608 7,0061 8,02E-13 2,45E e-5p 3,5677 3,9793 4,4367 3,5677 7,1608 7,0061 8,02E-13 2,45E f-5p 3,0188 3,4305 3,9336 3,0188 7,1608 7,0061 8,02E-13 2,45E a-3p 3,4762 4,1166 4,8484 3,4762 7,1608 7,0061 8,02E-13 2,45E a-5p 3,8421 4,2080 4,5740 3,8421 7,1608 7,0061 8,02E-13 2,45E b-3p 3,4762 3,9793 4,5282 3,4762 7,1608 7,0061 8,02E-13 2,45E b-5p 3,5219 3,9336 4,3453 3,5219 7,1608 7,0061 8,02E-13 2,45E c-3p 2,1498 2,4699 2,7901 2,1498 7,1608 7,0061 8,02E-13 2,45E c-5p 3,4762 3,9336 4,3453 3,4762 7,1608 7,0061 8,02E-13 2,45E d-5p 3,3390 3,8421 4,4367 3,3390 7,1608 7,0061 8,02E-13 2,45E a-3p 4,8027 5,3515 6,1291 4,8027 7,1608 7,0061 8,02E-13 2,45E a-5p 3,8421 4,1623 4,3910 3,8421 7,1608 7,0061 8,02E-13 2,45E b 3,4762 4,0251 4,6197 3,4762 7,1608 7,0061 8,02E-13 2,45E c-3p 3,5677 4,0708 4,6654 3,5677 7,1608 7,0061 8,02E-13 2,45E c-5p 2,7901 3,2475 3,7049 2,7901 7,1608 7,0061 8,02E-13 2,45E d-3p 3,2475 3,7049 4,1166 3,2475 7,1608 7,0061 8,02E-13 2,45E d-5p 2,4242 2,7901 3,1103 2,4242 7,1608 7,0061 8,02E-13 2,45E e 1,8753 2,3785 2,9273 1,8753 7,1608 7,0061 8,02E-13 2,45E f-3p 3,8421 4,2080 4,4825 3,8421 7,1608 7,0061 8,02E-13 2,45E f-5p 2,8816 3,6134 8,2331 2,8816 7,1608 7,0061 8,02E-13 2,45E g-3p 3,2018 3,7049 4,2080 3,2018 7,1608 7,0061 8,02E-13 2,45E g-5p 2,6986 3,2932 8,9650 2,6986 7,1608 7,0061 8,02E-13 2,45E h 2,6529 3,3847 8,4161 2,6529 7,1608 7,0061 8,02E-13 2,45E p 2,8359 3,2018 3,5219 2,8359 7,1608 7,0061 8,02E-13 2,45E-12

17 522-5p 3,4762 3,9336 4,3453 3,4762 7,1608 7,0061 8,02E-13 2,45E p 3,4762 3,8879 4,2995 3,4762 7,1608 7,0061 8,02E-13 2,45E p 2,3327 2,6529 2,9731 2,3327 7,1608 7,0061 8,02E-13 2,45E p 2,0583 2,3327 2,5614 2,0583 7,1608 7,0061 8,02E-13 2,45E a 3,0188 3,4305 3,7964 3,0188 7,1608 7,0061 8,02E-13 2,45E b-5p 1,7381 2,1040 2,3785 1,7381 7,1608 7,0061 8,02E-13 2,45E p 2,7444 3,7049 8,2331 2,7444 7,1608 7,0061 8,02E-13 2,45E p 4,7112 7,1354 9,3309 4,7112 7,1608 7,0061 8,02E-13 2,45E p 3,7049 6,4493 8,6448 3,7049 7,1608 7,0061 8,02E-13 2,45E p 3,6134 4,2538 9,6968 3,6134 7,1608 7,0061 8,02E-13 2,45E-12 99a-5p 4,5740 5,2601 6,0834 4,5740 7,1608 7,0061 8,02E-13 2,45E p 3,5677 6,2663 8,4161 3,5677 7,1608 7,0061 8,02E-13 2,45E-12 92a-1-5p 1,1892 1,6009 1,9668 1,1892 7,1608 7,0061 8,02E-13 2,45E p 3,4762 6,4035 8,5990 3,4762 7,1608 7,0061 8,02E-13 2,45E ,6592 5,9919 8,1874 3,6592 7,1608 7,0061 8,02E-13 2,45E p 2,1040 2,9273 4,0708 2,1040 7,1608 7,0061 8,02E-13 2,45E p 3,3847 6,0376 8,2331 3,3847 7,1608 7,0061 8,02E-13 2,45E ,3847 5,8547 8,0959 3,3847 7,1608 7,0061 8,02E-13 2,45E e-5p 1,9668 2,6529 3,7049 1,9668 7,1608 7,0061 8,02E-13 2,45E p 3,4305 5,8547 8,0502 3,4305 7,1608 7,0061 8,02E-13 2,45E p 3,4762 5,9004 8,0959 3,4762 7,1608 7,0061 8,02E-13 2,45E p 3,3390 5,7174 7,9587 3,3390 7,1608 7,0061 8,02E-13 2,45E e-3p 2,8816 6,4493 8,2331 2,8816 7,1608 7,0061 8,02E-13 2,45E f-3p 2,5157 7,8672 8,3703 2,5157 7,1608 7,0061 8,02E-13 2,45E p 3,2475 5,6260 7,8215 3,2475 7,1608 7,0061 8,02E-13 2,45E p 3,2475 5,6260 7,8215 3,2475 7,1608 7,0061 8,02E-13 2,45E-12 hsa-let-7a- 5p -5,6260-5,0771-4,5740-4,5740-7,1608-7,0061 8,02E-13 2,45E a-3p -2,6072-2,2870-2,0125-2,0125-7,1608-7,0061 8,02E-13 2,45E p -2,6986-2,2412-1,8296-1,8296-7,1608-7,0061 8,02E-13 2,45E a-5p -9,7883-4,8941-3,8421-3,8421-7,1608-7,0061 8,02E-13 2,45E a-3p -2,0583-1,8296-1,6466-1,6466-7,1608-7,0061 8,02E-13 2,45E a-3p -2,0583-1,6924-1,4179-1,4179-7,1608-7,0061 8,02E-13 2,45E p -8,4161-4,0708-3,3847-3,3847-7,1608-7,0061 8,02E-13 2,45E b-5p -3,8421-3,1103-2,5614-2,5614-7,1608-7,0061 8,02E-13 2,45E p -3,4762-3,1103-2,7901-2,7901-7,1608-7,0061 8,02E-13 2,45E p -8,6905-7,3641-3,8879-3,8879-7,1608-7,0061 8,02E-13 2,45E-12 20a-5p -2,1955-1,7838-1,4179-1,4179-7,1608-7,0061 8,02E-13 2,45E-12 20b-3p -7,9129-6,2663-3,7049-3,7049-7,1608-7,0061 8,02E-13 2,45E ,0044-6,6322-3,4762-3,4762-7,1608-7,0061 8,02E-13 2,45E p -3,0646-2,3785-1,8296-1,8296-7,1608-7,0061 8,02E-13 2,45E p -4,3910-3,9793-3,6134-3,6134-7,1608-7,0061 8,02E-13 2,45E p -3,4305-3,0188-2,6529-2,6529-7,1608-7,0061 8,02E-13 2,45E-12

18 301a-3p -4,0251-3,1103-2,5157-2,5157-7,1608-7,0061 8,02E-13 2,45E a-3p -2,5614-2,3327-2,1498-2,1498-7,1608-7,0061 8,02E-13 2,45E b-3p -2,2870-2,1040-1,9211-1,9211-7,1608-7,0061 8,02E-13 2,45E c-3p -4,2995-3,9336-3,6134-3,6134-7,1608-7,0061 8,02E-13 2,45E-12 30a-5p -3,8421-3,2018-2,6072-2,6072-7,1608-7,0061 8,02E-13 2,45E p -2,9731-2,6072-2,2870-2,2870-7,1608-7,0061 8,02E-13 2,45E p -1,4637-1,1892-1,0063-1,0063-7,1608-7,0061 8,02E-13 2,45E a-5p -4,0251-3,2018-2,6072-2,6072-7,1608-7,0061 8,02E-13 2,45E p -4,0251-3,0646-2,4699-2,4699-7,1608-7,0061 8,02E-13 2,45E p -3,3847-2,9273-2,5614-2,5614-7,1608-7,0061 8,02E-13 2,45E p -7,8215-2,9731-2,2870-2,2870-7,1608-7,0061 8,02E-13 2,45E-12 92b-3p -4,7569-4,4367-4,1623-4,1623-7,1608-7,0061 8,02E-13 2,45E-12 99b-5p -1,6009-1,2807-1,0063-1,0063-7,1608-7,0061 8,02E-13 2,45E p 3,1560 5,5802 7,7757 3,1560 7,1608 7,0061 8,02E-13 2,45E p 2,8816 5,5802 7,7300 2,8816 7,1608 7,0061 8,02E-13 2,45E d-3p -2,5614-2,3327-2,1498-2,1498-7,1608-7,0061 8,02E-13 2,45E p -3,4762-2,9731-2,5157-2,5157-7,1608-7,0061 8,02E-13 2,45E p 3,2018 5,5802 7,7757 3,2018 7,1608 7,0061 8,02E-13 2,45E p 3,0646 5,4430 7,6385 3,0646 7,1606 7,0061 8,03E-13 2,45E p -1,9211-1,4637-1,0977-1,0977-7,1605-7,0061 8,04E-13 2,45E p 1,1435 1,5551 2,0125 1,1435 7,1604 7,0061 8,04E-13 2,45E b-5p -7,9129-6,5408-2,6529-2,6529-7,1601-7,0061 8,06E-13 2,45E-12 15b-5p -2,4699-1,9211-1,4179-1,4179-7,1600-7,0061 8,07E-13 2,45E p -2,0583-1,6009-1,1892-1,1892-7,1598-7,0061 8,08E-13 2,45E a-3p -3,6134-2,6529-1,8753-1,8753-7,1598-7,0061 8,08E-13 2,45E b-3p 2,9731 5,6717 7,7757 2,9731 7,1577 7,0052 8,20E-13 2,47E p -2,7444-2,1040-1,5094-1,5094-7,1574-7,0052 8,22E-13 2,47E b-2-3p 2,8359 5,6260 8,0044 2,8359 7,1559 7,0049 8,31E-13 2,47E p -2,6986-2,0583-1,4637-1,4637-7,1557-7,0049 8,33E-13 2,47E p 2,9273 5,3058 7,5013 2,9273 7,1538 7,0037 8,44E-13 2,49E p -7,6842-6,0376-3,3847-3,3847-7,1512-7,0018 8,60E-13 2,53E p -1,7838-1,3722-1,0063-1,0063-7,1478-6,9991 8,82E-13 2,58E p 2,6072 5,7632 7,8215 2,6072 7,1412 6,9931 9,25E-13 2,69E p -3,0646-2,1955-1,5551-1,5551-7,1151-6,9671 1,12E-12 3,23E p 2,8359 5,5802 7,7300 2,8359 7,1123 6,9650 1,14E-12 3,28E p -1,6924-1,3264-0,9148-0,9148-7,0475-6,8996 1,82E-12 5,22E p 1,6009 2,3327 6,6780 1,6009 7,0179 6,8701 2,25E-12 6,42E p -2,8359-2,0583-1,4637-1,4637-6,9855-6,8378 2,84E-12 8,04E p -1,8753-1,4179-1,0063-1,0063-6,8934-6,7451 5,45E-12 1,53E p -7,5470-6,0376-3,1103-3,1103-6,8714-6,7235 6,36E-12 1,77E a-3p 2,5614 5,3058 7,4555 2,5614 6,8573 6,7098 7,02E-12 1,95E ,6466 2,2870 7,7757 1,6466 6,8509 6,7039 7,34E-12 2,03E-11

19 192-5p 0,9605 1,4637 1,9211 0,9605 6,8124 6,6653 9,60E-12 2,64E-11 27b-3p -1,4179-1,0520-0,7318-0,7318-6,7830-6,6360 1,18E-11 3,22E b-3p 2,4242 5,1228 7,5013 2,4242 6,7813 6,6350 1,19E-11 3,24E p 2,6529 5,0314 7,2269 2,6529 6,6899 6,5423 2,23E-11 6,06E a-5p -7,4098-5,7174-3,2018-3,2018-6,5853-6,4361 4,54E-11 1,23E b-3p 2,5157 5,3515 7,5470 2,5157 6,4535 6,3022 1,09E-10 2,94E d-3p 2,5157 4,9399 7,1354 2,5157 6,3833 6,2311 1,73E-10 4,63E p -7,9129-2,8359-2,0583-2,0583-6,3469-6,1946 2,20E-10 5,84E a-5p 2,5614 5,2601 7,4555 2,5614 6,3370 6,1852 2,34E-10 6,20E p -2,5157-1,8753-1,2807-1,2807-6,2656-6,1136 3,71E-10 9,74E p 2,5157 4,8941 7,0896 2,5157 6,2656 6,1136 3,71E-10 9,74E p -2,0125-1,5094-1,0520-1,0520-6,1493-5,9952 7,78E-10 2,03E p -6,3578-2,2870-1,5094-1,5094-6,1324-5,9787 8,66E-10 2,25E ,2412 4,7112 7,6385 2,2412 6,0799 5,9257 1,20E-09 3,11E ,1955-1,6009-1,0520-1,0520-5,7695-5,6079 7,95E-09 2,05E p 2,2870 4,8941 7,0439 2,2870 5,7285 5,5666 1,01E-08 2,60E p -2,7901-1,9211-1,2807-1,2807-5,7056-5,5438 1,16E-08 2,96E p -2,4699-1,7838-1,1892-1,1892-5,6721-5,5102 1,41E-08 3,58E p 2,2870 4,6654 6,8609 2,2870 5,5591 5,3957 2,71E-08 6,83E p 1,0977 1,8296 4,5740 1,0977 5,5254 5,3618 3,29E-08 8,24E p 2,3327 4,7112 6,9067 2,3327 5,5028 5,3394 3,74E-08 9,33E-08 10b-5p 1,1892 1,9668 6,6322 1,1892 5,4600 5,2961 4,76E-08 1,18E f-3p -7,1811-5,1228-2,5614-2,5614-5,3812-5,2157 7,40E-08 1,83E ,1811-5,5802-2,6529-2,6529-5,3541-5,1887 8,60E-08 2,12E-07 92a-2-5p -7,2726-5,5345-3,0188-3,0188-5,3224-5,1568 1,02E-07 2,51E p -2,3785-1,5551-0,9605-0,9605-5,1798-5,0115 2,22E-07 5,40E p -1,4179-1,0063-0,6404-0,6404-5,1616-4,9936 2,45E-07 5,93E p 2,1955 4,6197 6,8609 2,1955 5,1534 4,9859 2,56E-07 6,17E ,1498-1,4637-0,9148-0,9148-5,0496-4,8806 4,43E-07 1,06E c -1,8296-1,2807-0,7776-0,7776-5,0170-4,8477 5,25E-07 1,25E p -2,8359-1,8296-1,1435-1,1435-4,9719-4,8020 6,63E-07 1,57E ,0896-5,3973-2,7901-2,7901-4,9447-4,7756 7,63E-07 1,79E p 1,3722 2,1040 7,5013 1,3722 4,9365 4,7680 7,95E-07 1,86E ,1354-2,0583-1,2350-1,2350-4,9264-4,7583 8,38E-07 1,95E p -6,7237-2,1040-1,2807-1,2807-4,9113-4,7436 9,05E-07 2,10E p -2,1498-1,4179-0,8691-0,8691-4,8287-4,6603 1,37E-06 3,16E b-5p 0,7776 1,3722 2,0125 0,7776 4,8033 4,6353 1,56E-06 3,56E-06 30a-3p -6,8609-5,3058-2,2412-2,2412-4,7725-4,6052 1,82E-06 4,12E p 1,8753 4,2995 6,5408 1,8753 4,7603 4,5942 1,93E-06 4,34E p 0,5946 1,1435 1,6924 0,5946 4,7459 4,5802 2,08E-06 4,65E a -1,9211-1,3264-0,7318-0,7318-4,5955-4,4263 4,32E-06 9,59E p -6,9524-5,3973-2,4699-2,4699-4,5940-4,4256 4,35E-06 9,62E-06

20 524-3p 0,8691 1,6009 2,8816 0,8691 4,5911 4,4235 4,41E-06 9,71E b-5p -2,2412-1,5551-0,9148-0,9148-4,5401-4,3715 5,62E-06 1,23E p 1,7838 4,2080 6,4493 1,7838 4,4445 4,2733 8,81E-06 1,93E p 1,8296 4,2538 6,4950 1,8296 4,4098 4,2382 1,03E-05 2,25E a-2-3p -5,2143-1,8753-1,0063-1,0063-4,3808-4,2090 1,18E-05 2,57E b-5p -7,0896-2,1955-1,4637-1,4637-4,3675-4,1961 1,26E-05 2,72E a-5p 1,7381 4,2538 6,4493 1,7381 4,3219 4,1496 1,55E-05 3,33E p -1,6924-1,1435-0,6404-0,6404-4,3140-4,1423 1,60E-05 3,44E c-3p 0,9148 1,6924 6,9982 0,9148 4,2976 4,1261 1,73E-05 3,69E-05 23b-3p -1,5094-1,0063-0,5946-0,5946-4,2501-4,0777 2,14E-05 4,55E p -6,7695-5,0314-2,4699-2,4699-4,2481-4,0765 2,16E-05 4,57E b-5p -2,0125-1,3264-0,6861-0,6861-4,2459-4,0751 2,18E-05 4,60E b-3p -6,9067-5,1228-2,6072-2,6072-4,2187-4,0478 2,46E-05 5,17E p -6,7237-4,1623-1,7838-1,7838-4,1485-3,9757 3,35E-05 7,02E p -6,8609-5,1686-2,5614-2,5614-4,1395-3,9673 3,48E-05 7,27E p -2,5157-1,5551-0,8691-0,8691-4,1028-3,9300 4,08E-05 0, p 1,6009 4,3453 6,4950 1,6009 4,0254 3,8513 5,69E-05 0, ,5551 4,4825 6,5865 1,5551 4,0193 3,8459 5,84E-05 0, p 1,6466 4,0708 6,2663 1,6466 4,0070 3,8339 6,15E-05 0, a-5p 1,1892 4,3910 6,7695 1,1892 3,9566 3,7824 7,60E-05 0, p -6,7237-3,8421-1,4179-1,4179-3,9151-3,7400 9,04E-05 0, p -6,8152-5,0314-2,4699-2,4699-3,8962-3,7212 9,77E-05 0, p -2,4699-1,6466-0,8233-0,8233-3,8034-3,6254 0, , ,6322-4,8484-2,3327-2,3327-3,7396-3,5597 0, , p -1,6009-1,0063-0,5031-0,5031-3,7149-3,5349 0, , ,7237-4,9856-2,1955-2,1955-3,6412-3,4596 0, , p -6,7695-4,9856-2,4242-2,4242-3,6402-3,4596 0, , p -6,9067-2,2412-1,2807-1,2807-3,5932-3,4114 0, , p -6,9067-1,8296-0,9605-0,9605-3,5819-3,4005 0, , p 0,9148 2,0125 7,4555 0,9148 3,5749 3,3941 0, , p 1,3722 4,1166 6,2663 1,3722 3,5584 3,3777 0, , ,5031 1,3264 2,1498 0,5031 3,5333 3,3534 0, , p -2,2870-1,2807-0,5489-0,5489-3,4631-3,2819 0, , a-3p 0,5031 1,2807 2,2870 0,5031 3,4585 3,2780 0, , d -6,4035-1,8296-0,9605-0,9605-3,3988-3,2163 0, , a-5p -1,6466-1,0063-0,4574-0,4574-3,3489-3,1649 0, , p 1,4637 3,9793 6,2663 1,4637 3,3113 3,1265 0, , ,7237-3,3847-1,5551-1,5551-3,2719-3,0860 0, , b-3p 1,0977 3,9793 6,1748 1,0977 3,2266 3,0393 0, , d-5p -6,4493-3,3847-1,3722-1,3722-3,1648-2,9763 0, , p -2,0583-1,1892-0,5031-0,5031-3,1247-2,9350 0, , b-5p 0,7776 5,3973 6,5408 0,7776 3,1210 2,9322 0, ,00337

21 15a-5p -2,4699-1,3264-0,5031-0,5031-3,1013-2,9124 0, , p -6,6780-1,7381-0,7318-0,7318-3,0899-2,9025 0, , p -6,4493-4,7112-1,6466-1,6466-3,0865-2,8999 0, , ,7838-1,0977-0,4117-0,4117-3,0678-2,8812 0, , p 0,4117 1,1892 3,1560 0,4117 3,0639 2,8781 0, , p -1,7838-1,0063-0,3659-0,3659-3,0443-2,8585 0, , p -6,5408-4,7112-2,0583-2,0583-3,0017-2,8145 0, , p 0,6404 4,6654 6,5865 0,6404 2,9760 2,7884 0, , ,1955-1,1435-0,4117-0,4117-2,9624-2,7751 0, , p -6,1748-1,4637-0,5489-0,5489-2,9557-2,7691 0, , p -6,6780-4,8484-2,1498-2,1498-2,9469-2,7608 0, , ,4035-4,5740-1,9668-1,9668-2,9394-2,7539 0, , ,5865-1,4179-0,5489-0,5489-2,9205-2,7350 0, , a-3p -6,6780-1,4637-0,5489-0,5489-2,9110-2,7269 0, , a-3p 0,8691 3,7506 5,9919 0,8691 2,9109 2,7269 0, , ,9211-1,0520-0,3659-0,3659-2,9034-2,7201 0, , p -6,3578-1,6466-0,6861-0,6861-2,8494-2,6645 0, , p -6,4950-4,7112-1,9211-1,9211-2,8488-2,6645 0, , p 1,0977 3,6592 5,9461 1,0977 2,8315 2,6473 0, , ,8753-1,0520-0,3202-0,3202-2,7927-2,6087 0, , p -2,1955-1,1892-0,4117-0,4117-2,7921-2,6087 0, , b-3p 0,4117 1,2807 6,5865 0,4117 2,7767 2,5935 0, , a-3p -5,4887-1,2350-0,4117-0,4117-2,7747-2,5925 0, , p -6,3578-4,4825-0,7776-0,7776-2,7565-2,5742 0, , s 0,9605 3,8879 6,1748 0,9605 2,7124 2,5308 0, , p -6,2206-4,4825-1,4179-1,4179-2,6414-2,4566 0, , p -6,3578-4,5740-1,8296-1,8296-2,6393-2,4554 0, , ,8359-1,8296-0,4574-0,4574-2,6135-2,4310 0, , p 0,5946 4,0251 6,0834 0,5946 2,5724 2,3888 0, , k -6,1291-1,1892-0,2744-0,2744-2,4714-2,2848 0, , a-3p 0,7776 3,7049 6,0376 0,7776 2,4275 2,2392 0, , a-2-3p 0,5946 3,6592 5,8089 0,5946 2,3877 2,2001 0, , p -6,3578-1,3722-0,3659-0,3659-2,3735-2,1861 0, , p -6,1748-4,3453-0,6404-0,6404-2,3637-2,1768 0, , p -6,0834-1,0977-0,1830-0,1830-2,2842-2,0966 0, , p -2,2870-1,0977-0,1830-0,1830-2,2357-2,0471 0, , p -3,0646-1,1892-0,1830-0,1830-2,2044-2,0148 0, , p -6,0834-1,3264-0,2287-0,2287-2,2011-2,0124 0, , ,2663-1,4637-0,2744-0,2744-2,1739-1,9844 0, , a 0,1372 2,6986 5,9461 0,1372 2,1572 1,9677 0, , p 0,6861 3,7506 5,9919 0,6861 2,1525 1,9639 0, ,04954

22 Supplementary Table 2. List of used oligonucleotides and primer sequences, with their respective modifications. Oligo name Sequence and modification 3 Adaptor rapptggaattctcgggtgccaagg-ddc 1 5 adaptor NH2-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrG rarurcrhrhrhrhrhrhrhrhrcra 2 RT primer biotin-ccttggcacccgagaattccra rrna mask ATCGGCAAGCGACGCTCAGACAGGCGTAGCCCCGGGAGGAACCCGG GGCCGCAAGTGCGTTCGAAGTGTCGATGAT-TEG-biotin 3 RP1 AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCC GA INDEX-RPI1 INDEX-RPI2 INDEX-RPI3 INDEX-RPI4 INDEX-RPI5 INDEX-RPI6 INDEX-RPI7 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCCTTG

23 INDEX-RPI8 INDEX-RPI9 INDEX-RPI10 INDEX-RPI11 INDEX-RPI12 INDEX-RPI13 INDEX-RPI14 INDEX-RPI15 INDEX-RPI16 INDEX-RPI17 INDEX-RPI18 INDEX-RPI19 INDEX-RPI20 INDEX-RPI21 INDEX-RPI22 CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCCTTG

24 INDEX-RPI23 INDEX-RPI24 INDEX-RPI25 INDEX-RPI26 INDEX-RPI27 INDEX-RPI28 INDEX-RPI29 INDEX-RPI30 INDEX-RPI31 INDEX-RPI32 INDEX-RPI33 INDEX-RPI34 INDEX-RPI35 INDEX-RPI36 INDEX-RPI37 CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCCGGTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATATCGTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCGCCTGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCCATGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATAAAATGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTGTTGGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATATTCCGGTGACTGGAGTTCCTTG

25 INDEX-RPI38 INDEX-RPI39 INDEX-RPI40 INDEX-RPI41 INDEX-RPI42 INDEX-RPI43 INDEX-RPI44 INDEX-RPI45 INDEX-RPI46 INDEX-RPI47 INDEX-RPI48 CAAGCAGAAGACGGCATACGAGATAGCTAGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGTATAGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTCTGAGGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGTCGTCGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCGATTAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGCTGTAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATGAATGAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTCGGGAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATCTTCGAGTGACTGGAGTTCCTTG CAAGCAGAAGACGGCATACGAGATTGCCGAGTGACTGGAGTTCCTTG 1) rapp stands for 5 adenylation and ddc is a dideoxycytidine at the 3 end. 2) NH2 stands for amino linker and rn is ribo-nucleotides. rh stands for the combination of three ribo-nucleotides of ra, ru and rc. Eight rh ribo-nucleotides were designed to provide UMIs (Unique Mulecular Identifier). Sequence of rcra was used as spacer between UMI and small RNA sequences. 3) biotin and TEG-biotin stands for attached biotin group that can have TEG spacer.

26 Supplementary Table 3. Sequencing statistics of single-cell small RNA libraries. Details on the sequence library characteristics and the mapping statistics from aligning the libraries towards the human genome (assembly hg38) using STAR (see methods for the exact parameters used). Most analyses and figures of the study were based on the sequence libraries for which we sequenced a 41 bp RNA fragment (listed on top) but we also initially generated libraries wirth only 33 nt RNA fragments (listed below 41 bp single-cell data). Furthest down are details on the cell population-based libraries both using our protocol and using the commercial NEBNext protocol. Cells marked in red are the cells that were used in Figures 1a,b,c,d,e,f,g,j,k. For Figures 1h, i and in Supplementary Figures 4 and 5 all single-cell data was used (of both 33 and 41 bp inserts). A minority of single-cell data was generated without using the rrna masking strategy and these are denoted in the rrna mask column. Provided as an excel file: Supplementary Table 3.xlsx Supplementary Software. Python scripts for processing the small-rna sequence library data. Please also see our Github repository ( for further improved code. Provided as a compressed Zip archive: smallseq-master.zip