PIP-seq. Cells. Permanganate ChIP-Seq

Transcription

1 PIP-seq ells Formaldehyde Permanganate 5 Harvest Lyse Sonicate First dapter Ligation hip Elute Reverse rosslinks Piperidine cleavage Primer Extension Second dapter Ligation Deep Sequencing Permanganate hip-seq Supplementary Figure. PIP-seq protocol schema Shown is an illustration of the PIP-seq assay protocol as previously descried. In short, cells are cross-linked with formaldehyde and treated with potassium permanganate. hey are then lysed and sonicated to fragment sizes etween 2-5 p. n Illumina-compatile adapter is then ligated to the ends after end-repair and -tailing. he sample is then eluted from the antiody and crosslinks are reversed. Piperidine is then used to cleave oxidized thymines. Strands are denatured and a primer is annealed to the adapters, followed y second-strand synthesis (primer extension). second adapter is susequently ligated to the piperidine-cleaved end, via -tailing. he resulting lirary is then quantified and sequenced.

2 SS nucleotide frequency 26,63 Human SS FIIB op 25% Bottom 25%.4.4 solute Frequency solute Frequency c Log 2 ratio (normalized tag count) ll peaks (,) Pass threshold (95) Fail threshold (49) hip-exo PIP-seq RO-cap Supplementary Figure 2. Nucleotide frequencies surrounding annotated human SS (a) Heatmaps of nucleotide frequency within p of annotated SSs (N=26,63). Plots are sorted y FIIB PIP-seq tag counts located within 25 p of each SS. Only those tag 5 ends that mapped just 3 to a were counted, when sorting. () omposite plots from panel (a), separated out into the top 25% of FIIB PIP-seq peak tag counts versus the ottom 25%. (c) Log 2 ratio of the average called peak score for over the average random peak score for FIIB hip-exo, FIIB PIP-seq, and RO-cap

3 - position 5 end ag Reference genome FIIB PIP-seq c 5 Nucleotide occurence 26,63 Human SS 26,63 Human SS FIIB hip-exo d Relative Frequency Relative Frequency Normalized U.5 FIIB PIP-seq : FIIB hip-exo :. :.92 PIP-seq Nucleotide U :.48 :.4 :.2 :.9 :.69 hip-exo Supplementary Figure 3. FIIB PIP-seq validation (a) Schematic showing how reads were filtered y the specific - nucleotide 5 the to aligned sequence read () Heatmap distriution of FIIB PIP-seq (top) or hip-exo (ottom) tag 5 ends relative to all annotated human SSs 2, and separated out y the type of nucleotide (,,, ) occurring nucleotide upstream from the 5 end (i.e., - position). Plots are sorted y FIIB PIP-seq tag counts (that also have a - ) within 25 p of each SS. (c) omposite (average) of panel (a). Relative peak height values (mode) for each composite are reported. (d) rea under the curve (U) from panel normalized y the local nucleotide content, calculated as Σ(vg reads ±25 p window around SS) / (vg nucleotide frequency ±25 p window around SS)

4 - position 5 end ag Reference genome Pol II PIP-seq c 5 Nucleotide occurence 26,63 Human SS 26,63 Human SS Pol II hip-exo d Relative Frequency Relative Frequency Normalized U PIP-seq Pol II PIP-seq :. Pol II hip-exo :. : Nucleotide U :.49 :.39 :.7 :.45 :.42 hip-exo Supplementary Figure 4. Pol II PIP-seq validation (a-d) Same as Supplemental Figure 3, except the analysis was on Pol II instead of FIIB.

5 a FIIB FIIB signal Forward 25 Motif Occurence Reverse.5 Nucleosome Distance from FIIB PIP-seq peak (p) Motif Occurrence Nucleosome Randomized MNase-seq FIIB PIP-seq peaks (N=8,34) 25 RO-cap & MNase-seq signal Relative Motif Occurence FIIB PIP-seq peaks (N=8,34) Distance from FIIB PIP-seq peak (p) Motif Occurrence RO-cap PIP-seq Pol II Randomized RO-cap/Motif FIIB PIP-seq peaks (N=8,34) FIIB Distance from FIIB PIP-seq peak (p) Supplementary Figure 5. Motif enrichment relative to PI (a) omposite plots of FIIB PIP-seq, RO-cap, MNase-seq, and the enriched motif occurrence count and heatmaps of MNase-seq and enriched motif occurrence count relative to called FIIB PIP-seq peaks. JSPR 26 verterate motifs were scanned using FIMO in a 2 k window relative to FIIB PIP-seq peaks. ontrol data was generated y randomizing the sequence from each 2 k window. alled motifs were aligned relative to called peaks and sorted y distance of FIIB PIP-seq peak to called + nucleosome. () Heatmaps of PIP-seq data displaying the overlap of the motif occurrence and the RO-cap data. he lack line is the location of the called + nucleosome dyad and the grey line is exactly 73 p upstream representing the upper edge of the nucleosome.

6 Pol II PIP-seq ( / ) ( / ) 5 Nucleotide ratio FIIB PIP-seq 8 4 ( / ) ( / ) Pol II hip-exo FIIB hip-exo 8 Nucleotide ratio 5 ( / ) 4 ( / ) ( / ) ( / ) Supplementary Figure 6. PIP-seq strand normalization validation (a) Pol II and FIIB PIP-seq - tag counts were aligned relative to annotated human SSs (N=26,63), smoothed with a 7 p sliding window average, and then locally normalized (divided) y - tag counts treated similarly (/) so as to report on the relative -reactivity relative to the equally occurring nucleotide, as a readout of single-stranded DN. Similar control plots were made for and tags (/). () Pol II and FIIB hip-exo - N tags were normalized similarly as panel a.

7 c Proportion mrn Promoter ncrn Enhancer Insulator ranscription hromhmm states Repressed Heterochromatin O Biological Process mrn FIIB m RN m etaolic process translation nuclear-transcried m RN cataolic process m RN cataolic process translational initiation RN cataolic process translational term ination nuclear-transcried m RN cataolic process, nonsense-m ediated decay viral transcription viral gene expression Distal ncrn FIIB negative regulation of cytokine production regulation of adaptive immune response intrinsic apoptotic signaling pathway Fc-gamma receptor signaling pathway involved in phagocytosis response to type I interferon JK-S cascade regulation of D4-positive, alpha-eta cell differentiation negative regulation of stem cell differentiation growth horm one receptor signaling pathway regulat ion of D4-positive, alpha-eta cell activation p3 Occupancy 5 25 ncrn mrn Distance from FIIB (p) log(binom ial p value) Supplementary Figure 7. ncrn and mrn FIIB meta-analysis (a) Proportion of predicted regulatory functions ased on chromatin state maps for mrn and ncrn FIIB locations 3. () omposite plot of ENODE p3 hip-seq occupancy plotted relative to mrn and ncrn FIIB PIP-seq peaks 4. (c) O enrichment of closest annotated genes for mrn and distal ncrn FIIB locations 5.

8 Supplementary ale : Sequencing Experiment Statistics!!! ssay! Replicate! otal!ags! otal!''!ags!! Pol$II$ PIP&seq$ $ $62,98,427$$ $26,593,623$$ Pol$II$ PIP&seq$ 2$ $,845,45$$ $4,949,54$$ FIIB$ PIP&seq$ $ $43,354,58$$ $5,486,376$$ FIIB$ PIP&seq$ 2$ $8,579,59$$ $7,258,22$$ FIIB$ PIP&seq$ 3$ $2,444,57$$ $4,89,$$ Input$ PIP&seq$ $ $4,23,956$$ $,39,29$$ Pol$II$ hip&exo$ $ $9,743,244$$ $&$$ FIIB$ hip&exo$ $ $4,723,536$$ $&$$

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