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| Status |
Public on May 13, 2021 |
| Title |
HiChIP SMC1A A673 STAG2 WT Replicate 1 |
| Sample type |
SRA |
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| Source name |
A673 clones with STAG2 wild type
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A673
cell type: Ewing sarcoma cell line
genotype/variation: STAG2 wild-type (sgNT)
chip antibody: SMC1A (Bethyl A300-055A)
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| Treatment protocol |
The SMC1A antibody used in the ChIP experiments was by Bethyl A300-055A.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HiChIP was performed on the A673 clonal lines sgNT-1c4 and sgSTAG2-1c6 in duplicate based on a previously published protocol (Mumbach et al., 2016) with a few adaptations which were performed as previously described (Weintraub et al., 2017). The A673 clonal lines sgNT-1c4 and sgSTAG2-1c6 were tested with duplicates for each line crosslinked on separate occasions. The SMC1A antibody used in the ChIP experiments was by Bethyl A300-055A. Libraries were sequenced 100x100 on an Illumina Hi-Seq 2500 platform.
HiChIP was performed as described in (Mumbach et al., 2016) with a few modifications. Ten million cells were cross-linked for 10 min at room temperature with 1% formaldehyde in growth media and quenched in 0.125 M glycine. After washing twice with ice-cold PBS, the supernatant was aspirated and the cell pellet was flash-frozen in liquid nitrogen and stored at -80°C. Cross-linked cell pellets were thawed on ice, resuspended in 800 µL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% IGEPAL CA-630 with 1× cOmplete protease inhibitor (Roche, 11697498001)), and incubated at 4°C for 30 minutes with rotation. Nuclei were pelleted by centrifugation at 2500 rcf. for 5 min at 4°C and washed once with 500 µL of ice-cold Hi-C lysis buffer. After removing the supernatant, nuclei were resuspended in 100 µL of 0.5% SDS and incubated at 62°C for 10 minutes. SDS was quenched by adding 335 µL of 1.5% Triton X-100 and incubating for 15 minutes at 37°C. After the addition of 50 µL of 10X NEB Buffer 2 (NEB, B7002) and 375 U of MboI restriction enzyme (NEB, R0147), chromatin was digested at 37°C for 2 hours with rotation. Following digestion, MboI enzyme was heat-inactivated by incubating the nuclei at 62°C for 20 min. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52 µL of fill-in master mix, containing 37.5 µL of 0.4 mM biotin-dATP (Invitrogen, 19524016), 1.5 µL of 10 mM dCTP (Invitrogen, 18253013), 1.5 µL of 10 mM dGTP (Invitrogen, 18254011), 1.5 µL of 10 mM dTTP (Invitrogen, 18255018), and 10 µL of 5 U/µL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210), was added and the tubes were incubated at 37°C for 1 hour with rotation. Proximity ligation was performed by addition of 947 µL of ligation master mix, containing 150 µL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 µL of 10% Triton X-100, 7.5 µL of 20 mg/mL BSA (NEB, B9000), 10 µL of 400 U/µL T4 DNA ligase (NEB, M0202), and 655.5 µL of water, and incubation at room temperature for 4 hours with rotation. After proximity ligation, nuclei were pelleted by centrifugation at 2500 rcf. for 5 minutes and resuspended in 1 mL of ChIP sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS with protease inhibitor). Nuclei were sonicated using a Covaris S220 for 6 minutes with the following settings: fill level 8, duty cycle 5, peak incidence power 140, cycles per burst 200. Sonicated chromatin was clarified by centrifugation at 16,100 rcf. for 15 min at 4°C and supernatant was transferred to a tube. 60 µL of protein G magnetic beads were washed three times with sonication buffer, resuspended in 50 µL of sonication buffer. Washed beads were then added to the sonicated chromatin and incubated for 1 hour at 4°C with rotation. Beads were then separated on a magnetic stand and the supernatant was transferred to a new tube. 7.5 µg of SMC1A antibody (Bethyl A300-055A) was added to the tube and the tube was incubated overnight at 4°C with rotation. The next day, 60 µL of protein G magnetic beads were washed three time in 0.5% BSA in PBS and washed once with sonication buffer before being resuspended in 100 µL of sonication buffer and added to each sample tube. Samples were incubated for 2 hours at 4°C with rotation. Beads were then separated on a magnetic stand and washed three times with 1 mL of high salt sonication buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) followed by three times with 1 mL of LiCl wash buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS) and once with 1 mL of TE with salt (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 50 mM NaCl). Beads were then resuspended in 200 µL of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) and incubated at 65°C for 15 minutes to elute. To purify eluted DNA, RNA was degraded by the addition of 2.5 µL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 10 µL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 55°C for 45 minutes. Samples were then incubated at 65°C for 5 hours to reverse crosslinks. DNA was then purified using Zymo DNA Clean and Concentrate 5 columns (Zymo, D4013) according to the manufacturer’s protocol and eluted in 14 µL water. The amount of eluted DNA was quantified by Qubit dsDNA HS kit (Invitrogen, Q32854).
Tagmentation of ChIP DNA was performed using the Illumina Nextera DNA Library Prep Kit (Illumina, FC-121-1030). First, 5 µL of streptavidin C1 magnetic beads (Invitrogen, 65001) was washed with 1 mL of tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA pH 8.0, 1 M NaCl, 0.05% Tween-20) and resuspended in 10 µL of 2X biotin-binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 2 M NaCl). 54.19 ng purified DNA was added in a total volume of 10 µL of water to the beads and incubated at room temperature for 15 minutes with agitation every 5 minutes. After capture, beads were separated with a magnet and the supernatant was discarded. Beads were then washed twice with 500 µL of tween wash buffer, incubating at 55°C for 2 minutes with shaking for each wash. Beads were resuspended in 25 µL of Nextera Tagment DNA buffer. To tagment the captured DNA, 3.5 µL of Nextera Tagment DNA Enzyme 1 was added with 21.5 µL of Nextera Resuspension Buffer and samples were incubated at 55°C for 10 minutes with shaking. Beads were separated on a magnet and supernatant was discarded. Beads were washed with 500 µL of 50 mM EDTA at 50°C for 30 minutes, then washed three times with 500 µL of tween wash buffer at 55°C for 2 minutes each, and finally washed once with 500 µL of 10 mM Tris-HCl pH 7.5 for 1 minute at room temperature. Beads were separated on a magnet and supernatant was discarded. To generate the sequencing library, PCR amplification of the tagmented DNA was performed while the DNA is still bound to the beads. Beads were resuspended in 15 µL of Nextera PCR Master Mix, 5 µL of Nextera PCR Primer Cocktail, 5 µL of Nextera Index Primer 1, 5 µL of Nextera Index Primer 2, and 20 µL of water. DNA was amplified with 8 cycles of PCR. After PCR, beads were separated on a magnet and the supernatant containing the PCR amplified library was transferred to a new tube, purified using the Zymo DNA Clean and Concentrate-5 (Zymo D4003T) kit according to manufacturer’s protocol, and eluted in 14 µL water. Purified HiChIP libraries were size selected to 300-700 bp using a Sage Science Pippin Prep instrument according to manufacturer’s protocol and subject to paired-end sequencing on an Illumina HiSeq 2500. SMC1A libraries were initially sequenced with 100×100 bp paired-end sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
L22-2807_GT17-05036_TAAGGCGA_ATAGAGAG_S1
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| Data processing |
HiChIP raw reads were aligned to hg19 human reference genome using HiC-Pro v2.10 (Servant et al., 2015). Each of the four replicate samples was sequenced to a depth > 150M reads. Moreover, each sample passed stringent quality control with a minimum of 23% of all reads mapping to intrachromosomal loci. Replicate samples were merged. High-confidence loop calls were inferred on th emerged replicates using hichipper (Lareau and Aryee, 2018) using the union of SMC1A ChIP-Seq peaks from both the WT and STAG2 KO cell lines (144,863 genomic regions). Long range interactions spanning two anchor regions, termed DNA loops, were derived from linked paired-end reads that overlapped restriction fragments containing the SMC1A anchors. In total, 1,836,186 interactions spanning pairs of 102,398 genomic loci were observed among the four samples. As a majority of these interactions represent background proximity ligation, stringent filtering was performed to identify putative biologically functional DNA loops.
We called a set of 56,219 loops naive to any additional genetic or epigenetic annotation that met stringent criteria. These loops contained at least four reads with paired-end tags (PETs) in two or more replicates and were statistically-significant at a distance-dependent P-value of 1% and FDR of 1% based on the per-loop measures from the loop proximity bias correction algorithm originally implemented in Mango (Phanstiel et al., 2015). The set of 56,219 stringently filtered loops served as a basis for differential loop calling between the wild-type and STAG2 KO mutant cell lines. To identify loops with significant changes in contact coverage (either decrease or increase) in A673 with STAG2 KO compared to STAG2 WT cells, differential loops were called at using the diffloop (Lareau et al., 2018) package with the cutoffs |abs(fold change contact coverage)|≥2 and P-value ≤0.05.
Loop edges were annotated for their overlap with gene promoter regions and with ChIP-Seq binding sites for H3K27ac enhancers and EWS/FLI1. Loop were annotated for TSS+/-5kb promoter region landing for hg19 genes, and separately for expressed hg19 genes, in accordance with the 5kb resolution of our HiChIP data. Enhancer-Promoter loops were defined as the significant loops connecting a H3K27ac enhancer binding site on one anchor and a landing gene promoter (TSS+/-5kb region) on the opposite anchor. Enhancers were identified in the H3K27ac A673 ChIP-Seq data in this study as the H3K27ac binding sites outside the TSS+/-3kb regions. Loop anchors were fully annotated with the hosted enhancer binding sites and gene targets.
EWS/FLI1 status was assigned to the significant loops that host a EWS/FLI1 binding site (ChIP-Seq peak) on one anchor and a gene promoter (TSS+/-5kb) region on the opposite anchor. Thus, an EWS/FLI1 loop intermediates the "long interactions" between any of the EWS/FLI1 binding sites on one anchor to any of the gene promoter regions on the opposite anchor. For the purpose of the EWS/FLI1 loop annotation, we defined a consensus of 11,519 ChIP-Seq EWS/FLI1 binding sites on A673 cells by intersecting the peaks identified in the A673 EWS/FLI1 ChIP-Seq high coverage data in this study (macs2, FDR =0.01) with the union of the EWS/FLI1 peaks identified in two published A673 EWS/FLI1 ChIP-Seq studies (Riggi et al., 2014; Tomazou et al., 2015). Based on the consensus EWS/FLI1 peaks we identified 8,957 EWS/FLI1 loops. The anchors of the EWS/FLI1 loops were annotated with the EWS/FLI1 ChIP-Seq binding sites and gene promoter landing regions and with the STAG2 KO vs. WT differential status information for the host loop and for the gene. The fully annotated collection of all 23,580 EWS/FLI1 binding site – promoter long interactions harbored by the 8,957 EWS/FLI1 loops is available.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: (1) tab separated text: *.mango per replicate sample list of all unfiltered interactions as produced by hichipper. Shown: anchor1 genomic position, anchor2 genomic position, # PETs (reads) per sample, per-loop FDR measures from the loop proximity bias correction algorithm originally implemented in Mango (2) excel.xlsx: annotated list of all high confidence interactions (mango P < 0.01, mango FDR < 0.01). Legend provided. (3) excel xlsx: annotated list of all differential interactions (|diffloop fold change|> 2, difloop P-value < 0.05). Legend provided. (4) WashU Epigenome Browser long interaction files (bgzipped *.gz and indexed *gz.tbi format), separately for STAG2 WT and STAG2 KO samples, for the high confidence loops, respectively, for the differential (STAG2 KO vs. STAG2 WT) loops in the following categories: All, Enhancer-Promoter, EWS/FLI1, EWS/FLI1 & Enhancer-Promoter.
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| Submission date |
Feb 02, 2021 |
| Last update date |
Jul 17, 2021 |
| Contact name |
Gabriela Alexe |
| E-mail(s) |
galexe@broadinstitute.org
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| Organization name |
Broad Institute
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| Department |
Computational Biology and Bioinformatics
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| Street address |
415 Main St.
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE116495 |
The effect of STAG2 loss in Ewing sarcoma |
| GSE165977 |
The effect of STAG2 loss in Ewing sarcoma [HiChIP] |
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| Relations |
| BioSample |
SAMN17761271 |
| SRA |
SRX10004687 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5059681_HiChIP_SMC1A_A673_STAG2WT_1.unfiltered_interactions.all.mango.txt.gz |
9.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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