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| Status |
Public on Nov 03, 2022 |
| Title |
Scr3-RAD21 |
| Sample type |
SRA |
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| Source name |
HCT-116
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT-116
genotype: Parental
treatment: Control siRNA
chip antibody: RAD21, Abcam ab992
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| Treatment protocol |
RNAiMAX transfection reagent (Thermo Fisher Scientific) was diluted in Opti-MEM reduced serum medium (Thermo Fisher Scientific), and pipetted onto siRNA. Plates were incubated at room temperature for 20 minutes. HCT-116 cells were trypsinized and resuspended in antibiotic-free medium, then plated on siRNA duplexes to obtain a final siRNA concentration of 25 nM. After 72 h of RNAi treatment, cells were collected for experiments.
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| Growth protocol |
We obtained the HCT-116 cell line from ATCC (RRID:CVCL_0291). All cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin at 37 °C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: ChIP was performed based on (Shah et al., Dev Cell 2021). HCT-116 cells were cross-linked in culture medium by addition of methanol-free formaldehyde (ThermoFisher, final 1% v/v) and incubated at room temperature for 10 minutes with gentle nutation. Crosslinking was quenched by addition of glycine (final 125 mM) and incubated at room temperature for 5 minutes with gentle nutation. Media was aspirated and replaced with cold DPBS. Cells were scraped and transferred to conical tubes, then pelleted by centrifugation (1500 rpm, 3 minutes, room temperature). Pellets were flash frozen on liquid nitrogen and stored at -80C. For ChIP, 30 uL protein G magnetic beads (per sample; ThermoFisher) were washed three times in blocking solution (0.5% BSA in DPBS). Beads were then resuspended in 250 uL blocking solution and 2 ug antibody (RAD21, Abcam ab992; CTCF, Cell Signaling Technology 3418) was added. Beads and antibody were rotated at 4C for at least six hours. Nuclei were isolated from frozen cell pellets as follows: pellet was resuspended in 10 mL cold lysis buffer 1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors) and rotated at 4C for 10 minutes, followed by centrifugation (1500 rpm, 3 minutes, 4C). Supernatant was aspirated and the pellet was resuspended in 10 mL cold lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, and protease inhibitors) and rotated at room temperature for 10 minutes, followed by centrifugation (1500 rpm, 3 minutes, 4C). Supernatant was discarded and nuclei were resuspended in 1 mL cold lysis buffer 3 (10mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, and protease inhibitors) and transferred to pre-chilled 1mL Covaris AFA tubes (Covaris). Nuclei were sonicated using a Covaris S220 sonicator (high cell chromatin shearing for 15 minutes; Covaris). Sonicated chromatin was transferred to 1.5mL microcentrifuge tubes and Triton-X 100 was added (1% final v/v) followed by centrifugation (top speed, 10 minutes, 4C). Supernatant was transferred to a new tube. Antibody-conjugated protein G beads were washed three times in blocking solution, resuspended in 50 ul blocking buffer, and added to 500 ug sonicated chromatin. Chromatin was rotated overnight at 4C. 50 ug lysate was reserved in a separate tube at -20C for input. On day 2, beads were washed five times in 1 mL RIPA buffer (50mM HEPES-KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). Beads were then washed in 1 mL final wash buffer (1xTE, 50mM NaCl) for 2 minutes. Beads were finally resuspended in 210 uL elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS), and chromatin was eluted from beads by agitation at 65 °C for 30 minutes. 200 uL eluate was transferred to a new tube, and all samples (ChIP and input) were reverse cross-linked overnight at 65 °C with agitation for between 12 and 18 hours. 200 uL 1xTE was added to all samples, and samples were treated with RNase A (final 0.2mg/mL RNase; 37 °C for 2 hours) and proteinase K (final 0.2mg/mL Proteinase K; 55C for 2 hours). DNA was purified using phenol:chloroform extraction and resuspension in 10mM Tris-HCl pH 8.0.
ChIP-seq: ChIP and input DNA were quantified by Qubit (ThermoFisher) before library preparation using the NEBNext Ultra II DNA library prep kit (NEB). Samples were indexed for either single or dual-index sequencing. Library quality was assessed on Bioanalyzer (Agilent) and quantified by qPCR (Kapa Biosystems).
ChIP-seq: Quantified libraries were sequenced via Illumina sequencing using the NextSeq500 (single end, 75bp) platform. Data from the same libraries were merged for final analysis.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Scr3-RAD21
Scr2-RAD21.bed
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| Data processing |
ChIP-seq: Sequencing quality was examined using FASTQC to ensure that the library GC% and duplication rate were within expected range (v0.11.5). Reads were aligned against the hg38 reference genome using Bowtie2 (v2.2.5) allowing 1 mismatch in seed alignment ("-N 1") and soft clipping ("--local"). For single-end libraries, the primary alignment of each read with a MAPQ score higher than 10 was retained using SAMtools (v0.1.19). For paired-end reads, only properly paired primary alignment were retained and the maximum fragment size was set to be 2kb. Alignments were then filtered for PCR duplicates using SAMtools. Alignments that mapped to mitochondria, random contigs and ENCODE blacklisted regions were also removed for downstream analysis using BEDtools (v2.27.1).
ChIP-seq:Visualization tracks were generated using BEDtools, in which process, samples were normalized to 1M reads per library and corresponding input controls were subtracted from IP. Peaks were called for IP libraries against their corresponding input controls using MACS2 (v2.1.1), with default parameters and a 0.01 q-value cutoff. Finally, a non-overlapping union set of peaks was created by merging peaks in all samples using BEDtools such that all peaks that overlap by at least 1bp were merged. The union peaks were annotated to nearest genes using HOMER tools. ChIP-seq signal was quantified in each sample over each of the union peaks using Bwtools.
Assembly: hg38
Supplementary files format and content: ChIP-seq: Bigwig files were generated using BEDtools (v2.27.1) on each biological replicate; bed files of peaks for each biological replicate were called with MACS2 (v2.1.1), with default parameters and a 0.01 q-value cutoff
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| Submission date |
Nov 03, 2022 |
| Last update date |
Nov 05, 2022 |
| Contact name |
Jennifer Mary Luppino |
| E-mail(s) |
jennifm@pennmedicine.upenn.edu
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| Organization name |
University of Pennsylvania
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| Department |
Genetics
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| Lab |
Eric Joyce
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| Street address |
415 Curie Blvd., Room: 570
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104-6145 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE200773 |
Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression |
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| Relations |
| BioSample |
SAMN31593828 |
| SRA |
SRX18159173 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6709891_Scr3-RAD21.subtract.bw |
420.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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