SUPPLEMENL MERILS Eh-seq: RISPR epitope tagging hip-seq of DN-binding proteins Daniel Savic, E. hristopher Partridge, Kimberly M. Newberry, Sophia. Smith, Sarah K. Meadows, rian S. Roberts, Mark Mackiewicz, Eric M. Mendenhall, and Richard M. Myers Hudsonlpha Institute for iotechnology, Huntsville, L, US University of labama at Huntsville, Huntsville, L, US LE OF ONENS Supplemental able Legends... Page -3 Supplemental Figure Legends... Page 4- Supplemental able. Page Supplemental Figure Page 3 Supplemental Figure Page 4 Supplemental Figure 3 Page 5 Supplemental Figure 4 Page 6 Supplemental Figure 5 Page 7 Supplemental Figure 6 Page 8 Supplemental Figure 7 Page 9 Supplemental Figure 8 Page 0 Supplemental Figure 9 Page Supplemental Figure 0.. Page Supplemental Figure.. Page 3 Supplemental Figure.. Page 4 Supplemental Figure 3.. Page 5 Supplemental Figure 4.. Page 6 Supplemental Figure 5.. Page 7 Supplemental Figure 6.. Page 8 Supplemental Figure 7.. Page 9 Supplemental Figure 8.. Page 30 Supplemental Figure 9.. Page 3 Supplemental Figure 0.. Page 3 Supplemental Figure.. Page 33 Supplemental Figure.. Page 34 Supplemental Figure 3.. Page 35!!
Supplemental able. ene expression of transcription factors in Hep and MF7 cells. RPKM values for tested transcription factors in Hep and MF7 cells are displayed. Supplemental able. pfeh homology arm and grn designs. summary of all grn sequences and homology arm sequences for all DN-binding proteins and across all DNbinding proteins is given. For grns, the species, number of grns used, sequence of all grns, on-target (Doench et al. 04) scores and off-target (Hsu et al. 03) scores are shown. For 5-prime and 3-prime homology arms, the size of each arm, the type of homology arm (how it was generated; PR or glock) and the entire sequences are given. he DN sequences that are deleted, their number, including notes, as well as synonymous variants added to 5-prime homology arm at exonic F sequences spanning the grn site to prevent as9 re-cleavage events. Supplemental able 3. PR primer sequences. Primer sequences for cloning of both 5- prime and 3-prime homology arms are shown. Sequences in lowercase denote pfeh plasmid sequences required for cloning (ibson ssembly). Homology arms generated through glock synthesis are also denoted. PR validation primers are further given for all DN binding proteins tested. Supplemental able 4. Validation of cloned PR sequences spanning 5-prime and 3- prime homologous recombination sites. he raw Sanger sequence data for cloned PR fragments spanning the 5-prime (Endogenous sequence - FL tag) and 3-prime (Neomycin - Endogenous sequence) homologous recombination sites for all DN-binding proteins is shown. Sequences mapping to the endogenous locus are shown in black while sequences mapping to the pfeh plasmid are shown in blue.!!
Supplemental able 5. hip-seq data summary. summary of all hip-seq experiments is given, including (from left to right) the cell line, the hip-seq experiment name, antibody used, the number of aligned reads, the number of binding sites identified, normalized strand coefficients (NS), relative strand correlations (RS), quality scores, the number (Number) and percent (%) of peaks that are shared between technical Eh-seq replicates (ech Rep), the number (Number) and percent (%) of peaks that are shared between biological Eh-seq replicates (io Rep), the number (Number) and percent (%) of peaks shared between Ehseq and standard hip-seq replicate experiments using transcription factor antibodies (W Rep and W Rep) and the number (Number) and percent (%) of peaks that are shared between Eh-seq experiments and RISPR-modified hip-seq replicate experiments using transcription factor antibodies (Modified).! 3!
Supplemental Figure. PR validation of homologous recombination. PR validation of RISPR-mediated homologous recombination for all Hep and MF7 experiments. () schematic of primer locations at transcription factor genetic loci is given. elow, 5 - and 3 -end homologous recombination predicted PR band sizes are displayed. (-H) el images for all validations are given. From left to right, the gel images display ladder, 5 end and 3 end homology PR products. Names of each transcription factor and biological replicate (Rep and Rep) are shown above each gel. and sizes are given at the left of each gel and asterisks mark the synonymous band sizes across rows of gels. Hep results are given in ()-(F), while MF7 RD validation data is displayed in () and (H). Supplemental Figure. Western blot validation of FL tagged transcription factors. Protein validation experiments are displayed. Western blot images in ()-() and IP western blot images in (D) and (E) are given for all Hep and MF7 experiments (cell line denoted below western image). From left to right, westerns show protein size ladders, experimental and control western experiments. Red arrows mark the location of relevant bands. () Hep RD validation using FL antibody (FL) and RD antibody (RD) in RISPRmodified cell lines. ~30kDa band, the predicted size of full-length RD, is seen using both antibodies. () MF7 RD validation using FL antibody (FL) in RISPR-modified MF7 cell lines and FL antibody in wild-type MF7 cells (W FL). ~30kDa band is only observed in the RISPR-modified MF7 cells. () Hep P validation using FL antibody (FL) and P antibody (P) in RISPR-modified cell lines. Several bands ~50-60 kda in line with P protein sizes are visible using both antibodies. (D) Hep RE IP western validation using FL antibody (FL IP) or Ig (Ig IP) for IP pulldown followed by FL antibody blotting. ~37kDa and ~46KDa bands, sizes identified for the RE protein, are only observed in the FL IP pulldown experiment. (E) Hep F IP western validation using FL antibody (FL IP) or Ig (Ig IP) IP pulldown followed by FL antibody blotting. ~40kDa band, the predicted size of F, is only observed in the FL IP pulldown experiment.! 4!
Supplemental Figure 3. RD Sanger sequencing of PR amplicons representing homologous recombination site in Hep cells. () he endogenous sequence of the 3- prime end of RD targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram trace of PR amplicons spanning 3-prime homologous recombination sites. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graph below trace. Supplemental Figure 4. RE Sanger sequencing of PR amplicons representing homologous recombination site in Hep cells. () he endogenous sequence of the 3- prime end of RE targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram traces of PR amplicons spanning 3-prime homologous recombination sites. wo replicate transfection experiments are shown. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graphs below traces. Supplemental Figure 5. F Sanger sequencing of PR amplicons representing homologous recombination site in Hep cells. () he endogenous sequence of the 3- prime end of F targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram trace of PR amplicons spanning 3-prime homologous recombination sites. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graph below trace.! 5!
Supplemental Figure 6. P Sanger sequencing of PR amplicons representing homologous recombination site in Hep cells. () he endogenous sequence of the 3- prime end of P targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram trace of PR amplicons spanning 3-prime homologous recombination sites. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graph below trace. Supplemental Figure 7. nalysis of RISPR/as9 genome editing disruptions at untagged alleles. () he fraction of alleles in the Hep polyclonal population that show insertions or deletions (indels, in red) compared to unmodified alleles (in blue). Data is shown for F, RD and RE. () Examples of the low frequency sequence disruptions at the F coding sequence are shown. n image of the grn orientation at the coding is shown above. he protospacer adjacent motif (PM) sequence is shown (in yellow) along with the putative nuclease cleavage site (red arrow). Examples of disruptions at amino acid sequences and their associated frequencies are shown below. Supplemental Figure 8. Hep transcription factor motifs. Enriched binding motifs for all Hep experiments are shown. he names of hip-seq experiments are given above each DN sequence motif. Eh-seq experiments are labeled as FL while hip-seq experiments using transcription factor antibodies in wild-type cells are labeled as W. iological replicates are labeled rep or rep for RE data. For F, technical replicates are labeled rep and rep.! 6!
Supplemental Figure 9. Hep FL tag hip-seq correlations. () Rank correlations of normalized sequence read counts between standard hip-seq replicates (W replicate and W replicate ) using transcription factor antibodies in wild-type Hep cells. () Rank correlations of normalized sequence read counts between standard hip-seq data (W) using transcription factor antibodies in wild-type Hep cells and Eh-seq experiments (FL) using FL antibodies in RISPR-modified Hep cells. Rank correlations in () are given. In (), average rank correlations for all FL and wild-type hip-seq replicate pairwise comparisons are displayed. ranscription factor names are given above each plot. Supplemental Figure 0. Hep Epitope tag hip-seq technical replicate correlations. Rank correlations of normalized sequence read counts between Eh-seq technical replicates (FL replicate and FL replicate ). Rank correlations are displayed in each plot while tagged transcription factor names are given above plots. Supplemental Figure. Hep RE hip-seq site correlations with FL tag biological replicate. () Rank correlations of normalized sequence read counts between a second Eh-seq biological replicate (FL iological rep ) and standard hip-seq data (W) using transcription factor antibodies in wild-type Hep cells. () Rank correlations of normalized sequence read counts between Eh-seq biological replicates (FL iological rep and FL iological rep ). verage rank correlations for all FL and/or wild-type hip-seq replicate pairwise comparisons are displayed on each plot.! 7!
Supplemental Figure. Hep correlations with RISPR-modified hip-seq data. () Rank correlations of normalized sequence read counts between a hip-seq experiments in RISPR-modified cells using transcription factor antibodies (Modified cells) and hip-seq data in wild-type Hep cells using transcription factor antibodies (W). () Rank correlations of normalized sequence read counts between hip-seq experiments in RISPR-modified cells using transcription factor antibodies (Modified cells) and Eh-seq experiments using FL antibodies in RISPR-modified cells (FL). verage rank correlations for all pairwise comparisons are shown. ranscription factor names are given above each plot. Supplemental Figure 3. Differentially regulated gene p-value Q-Q plots. Q-Q plot of observed versus expected p-values (-log0 transformed) of differentially regulated genes. Differentially regulated gene comparisons (using the DESeq program) of wild-type Hep RNseq biological replicate data (in black), as well as wild-type Hep RN-seq data versus RISPR-modified Hep cells tagging F (in blue) or tagging RD (in gray) are given. ll comparisons exhibit nearly identical patterns and an overall paucity of significant differentially regulated genes. Supplemental Figure 4. RN-seq read counts across DN-binding protein mrn transcripts. RN-derived read counts across RD () and F () transcripts are displayed. Reads mapping to epitope tag sequences are highlighted in red, suggesting a substantial proportion of RN molecules are tagged in polyclonal Hep cell populations.! 8!
Supplemental Figure 5. Sequence integrity across 5-prime homologous recombination sites on mrn. RN-derived reads spanning 5-prime homologous recombination sites are displayed for RD () and F () epitope tagged cells. t each endogenous nucleotide location upstream of the recombined pfeh construct, the number of reads matching to the reference genome (gray), or that contain mismatches (blue), insertions (red) and deletions (green) are shown. hese data support a low level of mis-targeting at the 3-prime ends of the coding regions of RD and F. Supplemental Figure 6. Sequence integrity across 3-prime homologous recombination sites on mrn. RN-derived reads spanning 3-prime homologous recombination sites are displayed for RD () and F () epitope tagged cells. t each endogenous nucleotide location downstream of the recombined pfeh construct, the number of reads matching to the reference genome (gray), or that contain mismatches (blue), insertions (red) and deletions (green) are shown. hese data support a low level of mis-targeting at the 3-prime ends of the coding regions and the beginning of the 3-prime untranslated regions of RD and F. Supplemental Figure 7. RD Sanger sequencing of PR amplicons representing homologous recombination site in MF7 cells. () he endogenous sequence of the 3- prime end of RD targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram traces of PR amplicons spanning 3-prime homologous recombination sites. wo replicate transfection experiments are shown. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graphs below traces.! 9!
Supplemental Figure 8. MF7 transcription factor motifs. Enriched binding motifs for all RD MF7 experiments. he names of RD hip-seq experiments are given above each motif. Data is shown for Eh-seq FL antibody biological replicate experiments and standard hip-seq experiments in wild-type MF7 cells using transcription factor antibodies. Supplemental Figure 9. MF7 RD FL tag hip-seq correlations. () Rank correlations of normalized sequence read counts between standard hip-seq replicates (W replicate and W replicate ) using transcription factor antibodies in wild-type MF7 cells. () Rank correlations of normalized sequence read counts between standard hip-seq data (W) using transcription factor antibodies in wild-type MF7 cells and Eh-seq experiments (FL) using FL antibodies in RISPR-modified MF7 cells. () Rank correlations of normalized sequence read counts between Eh-seq technical replicates (FL rep and FL rep ). In () and (), rank correlations are displayed. In (), average rank correlations for all FL and wild-type hip-seq replicate pairwise comparisons are given. Supplemental Figure 0. MF7 hip-seq site correlations with FL tag biological replicate. () Rank correlations of normalized sequence read counts between a second MF7 Eh-seq biological replicate (FL iological rep ) and standard hip-seq data (W) using transcription factor antibodies in wild-type MF7 cells. () Rank correlations of normalized sequence read counts between a Eh-seq biological replicates (FL iological rep and FL iological rep ) are given. he average rank correlations for all FL and wild-type hip-seq replicate pairwise comparisons are displayed.! 0!
Supplemental Figure. MF7 correlations with RISPR-modified hip-seq data. () Rank correlations of normalized sequence read counts between hip-seq experiments in RISPR-modified MF7 cells using transcription factor antibodies (Modified cells) and hipseq data in wild-type MF7 cells using transcription factor antibodies (W). () Rank correlations of normalized sequence read counts between hip-seq experiments in RISPRmodified MF7 cells using transcription factor antibodies (Modified cells) and Eh-seq experiments (FL) using FL antibody in RISPR-modified cells. verage rank correlations for all FL and wild-type hip-seq replicate pairwise comparisons in RISPR-modified cells are given.!! Supplemental Figure. P Sanger sequencing of PR amplicons representing homologous recombination site in mouse embryonic stem cells. () he endogenous sequence of the 3-prime end of P targeted for pfeh homologous recombination (blue area). () Schematic of the homologous recombination pfeh construct. Red arrows point to primers used for amplification of 3-prime homologous recombination sites. () Sanger sequencing electropherogram traces of PR amplicons spanning 3-prime homologous recombination sites. he 3-prime end of the pfeh construct is shown at the left, while the endogenous 3-prime un-translated region (UR) is shaded in gray. Quality scores indicating confidence at each base position are indicated by bar graphs below traces.!!!! Supplemental Figure 3. P Eh-seq experimentation summary in mouse embryonic stem cells. () he canonical P binding motif was identified and enriched at Eh-seq binding sites. In () and (), DN-binding protein read enrichment tracks for P Eh-seq experiments on the US enome rowser are shown at distinct genetic loci. In () Eh-seq data at two large loci are shown, while in () data for two smaller genome windows sizes are displayed.!!!!!!
ranscription factor Hep RPKM MF7 RPKM RD 40.09 37.55 RE.84 5.4 NRH 7.59 N/ F 5.99 5.53 P.4 N/ Supplemental able
FL P F Name NEO 5 and Size 3 and Size RD 363 78 RE 48 90 P 676 500 F 66 433 RE Rep 3kb kb.5kb.kb kb RD Rep 3kb kb.5kb.kb kb RD 3kb kb.5kb.kb kb DRE Rep E P * * * ** * * * ** F F * * * ** H RD Rep * * * ** Supplemental Figure 3
D RD (~30KDa) Hep E RD (~30KDa) MF7 P (~50-60KDa) Hep RE (~37&46KDa) Hep F (~40KDa) Hep Supplemental Figure 4
!"#$%& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 3 5
'!()%& '!()%& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 4 6
"*+%& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 5 7
,")-"& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 6 8
Supplemental Figure 7 9
3 4 5 6 7 4 5 6 7 8 9 bits 0 3 RD W 4 5 6 7 8 9 0 3 4 5 6 7 8 9 0 MEME (no SS) 7.03.5 3:58 bits RD Flag 0 3 4 5 6 7 8 9 0 3 MEME (no SS) 05.0.5 :37 4 5 RE W RE Flag rep RE Flag rep bits 0 3 4 5 6 7 8 9 0 3 4 5 MEME (no SS) 5.03. 0:46 bits 0 3 4 5 6 7 8 9 0 3 MEME (no SS) 06.03.5 3:46 4 5 bits 0 3 4 5 6 7 8 9 0 5 3 MEME (no SS) 07.04.5 05:37 4 bits 0 P W 3 4 5 6 7 8 9 0 3 4 5 MEME (no SS).03.4 08:56 P Flag bits 0 3 4 5 6 7 8 9 0 3 4 5 MEME (no SS).0.5 4:9 F Flag rep F Flag rep bits 0 8 9 0 3 4 5 MEME (no SS) 7.0.5 5:9 bits 0 3 0 3 4 6 MEME (no SS) 06.03.5 5:9 5 7 Supplemental Figure 8 0
RD RD! = 0.84! = 0.80 log (W replicate ) RE! = 0.93 log (Flag) RE! = 0.86 P P! = 0.94! = 0.90 log (W replicate ) log (W) Supplemental Figure 9
RD RE! = 0.97! = 0.9 log (Flag replicate ) P F! = 0.98! = 0.9 log (Flag replicate ) Supplemental Figure 0
log (Flag iological rep )! = 0.90 RE log (W) log (Flag iological rep )! = 0.89 RE log (Flag iological rep ) Supplemental Figure 3
RD RD! = 0.86! = 0.93 log (Modified cells) RE RE! = 0.78 log (Flag)! = 0.86 P P! = 0.93! = 0.95 log (W) log (Modified cells) Supplemental Figure 4
Supplemental Figure 3 5
RD reads 0 00 00 Exon 4 3' of ag ag Exon 4 5' of ag Exon 3 Exon Exon Exon 0 Exon 9 Exon 8 Exon 7 Exon 6 Exon 5 Exon 4 Exon 3 Exon Exon F reads 0 0 40 Exon 7 3' of ag ag Exon 7 5' of ag Exon 6 Exon 5 Exon 4 Exon 3 Exon Exon Supplemental Figure 4 6
RD event counts 00 80 60 40 Reference Mismatch Insertion Deletion 0 0-49 bp - 48 bp - 47 bp - 46 bp - 45 bp - 44 bp - 43 bp - 4 bp - 4 bp - 40 bp - 39 bp - 38 bp - 37 bp - 36 bp - 35 bp - 34 bp - 33 bp - 3 bp - 3 bp - 30 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp - 0 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp - 0 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp distance from 5' end of tag F event counts 5 Reference Mismatch Insertion Deletion 0 5 0-44 bp - 43 bp - 4 bp - 4 bp - 40 bp - 39 bp - 38 bp - 37 bp - 36 bp - 35 bp - 34 bp - 33 bp - 3 bp - 3 bp - 30 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp - 0 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp - 0 bp - 9 bp - 8 bp - 7 bp - 6 bp - 5 bp - 4 bp - 3 bp - bp - bp distance from 3' 5' end of tag Supplemental Figure 5 7
RD event counts 80 60 40 0 Reference Mismatch Insertion Deletion 0 + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 0 bp + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 0 bp + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 30 bp + 3 bp + 3 bp + 33 bp + 34 bp + 35 bp + 36 bp + 37 bp + 38 bp + 39 bp + 40 bp + 4 bp + 4 bp + 43 bp + 44 bp + 45 bp + 46 bp + 47 bp + 48 bp + 49 bp distance from 3' end of tag 8 F event counts 6 4 Reference Mismatch Insertion Deletion 0 + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 0 bp + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 0 bp + bp + bp + 3 bp + 4 bp + 5 bp + 6 bp + 7 bp + 8 bp + 9 bp + 30 bp + 3 bp + 3 bp + 33 bp + 34 bp + 35 bp + 36 bp + 37 bp + 38 bp + 39 bp + 40 bp + 4 bp + 4 bp + 43 bp + 44 bp + 45 bp + 46 bp distance from 3' end of tag Supplemental Figure 6 8
!"#$%& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 7 9
bits 0 3 4 RD W 5 6 7 8 0 9 3 4 5 6 7 8 9 0 3 MEME (no SS) 5.07. 9:59 RD Flag biological replicate bits 0 3 4 5 6 7 8 9 0 3 4 5 MEME (no SS) 05.0.5 :37 RD Flag biological replicate bits 0 3 4 5 6 7 8 9 0 3 4 5 6 7 8 9 MEME (no SS) 07.04.5 03:07 Supplemental Figure 8 30
log (W replicate )! = 0.95 log (W replicate )! = 0.89 log (W) log (Flag rep ) log (Flag)! = 0.94 log (Flag rep ) Supplemental Figure 9 3
log (Flag iological rep )! = 0.88 RD log (W) log (Flag iological rep )! = 0.86 RD log (Flag iological rep ) Supplemental Figure 0 3
log (Modified cells)! = 0.90 RD log (W)! = 0.85 RD log (Flag) log (Modified cells) Supplemental Figure 33
,./0.& HOM FL P NeoR HOM 3 End of pfeh construct Endogenous 3 UR Supplemental Figure 34
chr: kb 90,73,000 90,75,000 500bp chr: 30,093,000 30,093,500 5Mb chr: 78,000,000 83,000,000 88,000,000 00kb chr: 9,650,000 9,900,000 30,50,000 Supplemental Figure 3 35