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| Status |
Public on Oct 07, 2024 |
| Title |
NIPBL-D7-DMSO_RAD21_ChIP |
| Sample type |
SRA |
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| Source name |
hTERT RPE-1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: hTERT RPE-1
genotype: mGreenLantern-P2A-FKBP12(F36V)-NIPBL
clone name: NIPBL-D7
treatment: DMSO 24hr
antibody: RAD21 (Abcam #ab992)
spike-in: ~10% NIH/3T3
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| Treatment protocol |
Asynchronous cells were actively cycling when treated with DMSO or dTAGV-1 for the stated times.
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| Growth protocol |
hTERT RPE-1 were maintained in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin, and grown at 37°C with 5% CO2. NIH/3T3 cells (for spike-in) DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For HA ChIP-seq, cells were fixed with 2 mM DSG for 45 minutes, washed with DPBS, then fixed further 1% formaldehyde for 10 minutes. Formaldehyde was quenched with 125mM glycine for 10 minutes. For other ChIP-seq experiments, cells were fixed with 1% formaldehyde for 10 minutes, then quenched with 125mM glycine for 10 minutes. All cells were lysed using formaldehyde lysis buffer (FALB; 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with 1% SDS and 1X PIC, and sonicated for 9 minutes (10 seconds on/10 seconds off) at 25% power using an Active Motif Epishear Probe Sonicator. Antibodies were pre-bound to blocked Protein A Dynabeads for 6-8 hours at 4°C before being added to chromatin that had been diluted out 1:10 with FALB (no SDS), incuating overnight at 4°C. The following day, beads were washed as described above, then eluted for 1 hour at 65°C in 50 mM Tris pH 8, 1 mM EDTA, 1% SDS. Supernatant was transferred to a new tube, supplemented with 200 mM NaCl and proteinase K, then incubated for 30 minutes at 55°C followed by overnight at 65°C. The following day, samples were treated with RNAse for 2 hours at 37°C, before DNA was purified using Zymo ChIP DNA Clean & Concentrator kit (#D5205).
For CTCF input and ChIP, samples were size-selected using AMPure beads and libraries prepared using 2S Plus DNA Library Kit (IDT #10009878). Otherwise, libraries were prepared using Diagenode Microplex v3 (#C05010001) and UDIs (Set I: # C05010008; Set 2: #C05010009).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq X |
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| Data processing |
ChIP-seq data was aligned to the hg38 reference genome using BWA-MEM.
Aligned data was de-duplicated using Picard MarkDuplicates.
Low quality and supplementary reads, and improper pairs were filtered out using samtools.
Reads aligning only to the primary chromosomes (chr1-22/X/Y) were captured using samtools. If required, hg38-aligned reads were also separated out from mm10-aligned reads.
Blacklisted regions were removed using bedtools intersect.
deepTools bamCoverage was used to generate BigWig files.
MACS2 was used to call peaks from the ChIP sample, using the input sample as a control.
Assembly: hg38
Supplementary files format and content: BigWig
Supplementary files format and content: narrowPeak
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| Submission date |
Sep 21, 2024 |
| Last update date |
Oct 07, 2024 |
| Contact name |
Jesse R Dixon |
| E-mail(s) |
jedixon@salk.edu
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| Organization name |
Salk Institute for Biological Studies
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| Lab |
PBL-D
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| Street address |
10010 N. Torrey Pines Rd.
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL34281 |
| Series (2) |
| GSE277720 |
Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [ChIP-seq] |
| GSE277721 |
Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation |
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| Relations |
| BioSample |
SAMN43853007 |
| SRA |
SRX26159218 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8528494_NIPBL-D7-DMSO_RAD21_ChIP.bw |
130.2 Mb |
(ftp)(http) |
BW |
| GSM8528494_NIPBL-D7-DMSO_RAD21_ChIP.narrowPeak.gz |
1.1 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
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