|
| Status |
Public on Dec 19, 2013 |
| Title |
H3K4me3 HS Senescent ChipSeq biological replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Cultured fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: normal foreskin
chip antibody: 07-449 from Millipore
strain: Hs68
|
| Treatment protocol |
Fibroblasts (8×105 cells per 10 cm dish) were infected with packaged LKO lentivirus vector containg CBX7shRNA (Sigma NM_175709.1-153s1c1). Infected cells were selected in puromycin (0.5ug/ml) and harvested after 10-14 days
|
| Growth protocol |
The BF and Hs68 strains of human fibroblasts (30 population doublings) were cultured in Dulbecco modified Eagles medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 μ/ml streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chip-Seq: DNA samples were end repaired, poly-A tailed and Illumina single end adapters were ligated following the standard Illumina protocol with minor adjustments. Agencourt AMPure XP beads at 0.8x ratio were used to size select out adapter dimers after adapter ligation. The Illumina kit Phusion enzyme was replaced by Kapa HiFi HotStart ready mix. Post PCR, AMPure XP beads were used at a 1:1 ratio to maintain size integrity and to allow use of the Invitrogen SizeSelect E-gel system. Samples were finally purified with QAIquick gel extraction kit and quality controlled on the DNA 1000 BioAnalyser 2100 chip before clustering and subsequent 36bp single end sequencing on the GAIIx analyser.
RNA-Seq: Total RNA was extracted using the High Pure RNA extraction Kit (Roche). RNA samples were quality controlled (QC) using the 6000 Nano RNA Chip on the BioAnalyser 2100 (Agilent) and subjected to poly-A selection using Sera-Mag oligo dT beads (Thermo Fisher Scientific Inc.). Libraries were prepared using the Directional mRNA-Seq Library Prep. v1.0. Pre-Release Protocol from Illumina with minor adjustments. The Illumina kit Phusion enzyme was replaced by Kapa HiFi HotStart ready mix which reduced the overall volume of the PCR and the ratio for the Agencourt AMPure XP beads was adjusted accordingly. The standard PCR cycling was also changed to match the concentration of the total RNA from the initial QC. After passing the final QC, the libraries were subjected to cluster formation and then 72bp single end sequencing on the GAIIx analyser.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
ChipSeq: Duplicates were removed using the Picard MarkDuplicates program (picard-tools package version 1.48; http://picard.sourceforge.net) with default parameters.
RNASeq: Alignments were performed to the UCSC version of the human transcriptome (Illumina iGenomes resource) using RSEM (version 1.2.4; Li and Dewey 2011).
|
| |
|
| Submission date |
Dec 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gordon Peters |
| E-mail(s) |
gordon.peters@cancer.org.uk
|
| Phone |
+44 207 269 3049
|
| Organization name |
CRUK London Research Institute
|
| Lab |
Molecular Oncology
|
| Street address |
44 Lincolns Inn Fields
|
| City |
London |
| ZIP/Postal code |
WC2A 3LY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE40740 |
Genome wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts |
|
| Relations |
| BioSample |
SAMN02471458 |
| SRA |
SRX396591 |
