 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 19, 2015 |
| Title |
dCas9_KRAB_HS2_CR4_H3K9me3_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Cultured K562 cells_dCas9_KRAB_HS2_CR4_H3K9me3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
transduced gene: dCas9-KRAB
grna target: globin HS2 enhancer
chip antibody: anti-H3K9me3
chip antibody vendor: Abcam
chip antibody cat. #: ab8898
chip antibody lot #: GR188072-1
|
| Treatment protocol |
K562 cells were transduced with lentivirus to stably express dCas9 or dCas9-KRAB and HS2 targeted gRNA. To facilitate transduction, the cationic polymer polybrene was added at a concentration of 4 µg/mL to the viral media. The lentivirus contained a puromycin resistance gene, and 1 µg/ml puromycin was used to initiate selection for transduced cells approximately 96 hours after transduction.
|
| Growth protocol |
K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each replicate, 2 ×10^7 nuclei were re-suspended in 1 mL of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4). Samples were sonicated using a Diagenode Bioruptor XL sonicator at 4°C to fragment chromatin to 200-500 bp segments. Insoluble components were removed by centrifugation for 15 min at 15000 rpm. We conjugated 5 µg of anti-FLAG (Sigma-Aldrich, F1804), or anti-H3K9me3 (Abcam, ab8898) to 200 µl of either sheep anti-rabbit or sheep anti-mouse IgG magnetic beads (Life Technologies 11203D/11201D). Sheared chromatin in RIPA was then added to the antibody-conjugated beads and incubated on a rotator overnight at 4°C. After incubation, beads were washed five times with a LiCl wash buffer (100 mM Tris at pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), and remaining ions were removed with a single wash with 1 mL of TE (10 mM Tris-HCl at pH 7.5, 0.1 mM Na2EDTA) at 4°C. Chromatin and antibodies were eluted from beads by incubating for 1 h at 65°C in IP elution buffer (1% SDS, 0.1 M NaHCO3), followed by incubating overnight at 65°C to reverse formaldehyde cross-links. DNA was purified using MinElute DNA purification columns (Qiagen).
Illumina TruSeq adapted libraries were constructed using an Apollo 324 NGS Library Prep System with a PrepX Complete ILMN DNA Library Kit (WaferGen Biosystems Inc). ChIP products were amplified with 15 cycles of PCR, and fragments 150-700 bp in length were selected using an AxyPrep MAG PCR Clean-Up Kit (Axygen MAG-PCRCL-50). Libraries were sequenced using single end 50 bp reads on an Illumina HiSeq.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Reads aligning to lentiviral sequence were filtered out using Bowtie2 (v2.1.0) with default parameters.
Remaining reads were aligned to hg19 reference genome using Bowtie2 (v2.1.0) with default parameters.
Aligned reads were sorted using samtools (v1.2).
PCR Duplicates were removed using samtools (v1.2).
Reads aligning to ENCODE blacklist regions were removed using BEDTools (v2.23.0, intersectBed).
Read normalized (reads per million mapped reads) bedgraph files were made using BEDTools (v2.23.0, genomeCoverageBed).
BigWig files were made from bedgraph files using bedGraphToBigWig (v4).
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files containing RPM normalized read signal
|
| |
|
| Submission date |
Jul 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mudit Chaand |
| Organization name |
Syros Pharmaceuticals
|
| Street address |
35 Cambridgepark Drive
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02140 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE70671 |
Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements |
|
| Relations |
| BioSample |
SAMN03856384 |
| SRA |
SRX1092398 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1819887_KRAB_CR_4_2_H3K9me3.bw |
108.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
