|
| Status |
Public on Dec 03, 2012 |
| Title |
H3K27me3_1x10e5_R3 |
| Sample type |
SRA |
| |
|
| Source name |
Primary (live-frozen) CD4+ cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell number: 100000
chip antibody: H3K27me3 (Upstate Biotech, Cat# 07-449)
|
| Growth protocol |
Both cultured and primary CD4+ and CD8+ lymphocytes used. CD8+ and CD4+ cells were sequentially isolated using an AutoMACS Pro separator (Miltenyi Biotec, Köln, Germany) by positive and negative isolation, respectively. Cultured cells were activated using Human T-Expander CD3/CD28 (Invitrogen, Carlsbad, CA) according to manufacturers instructions and cells were cultured in X-VIVO media (Lonza) supplemented with Interleukin II (10 ng/ul) for a period of two weeks at 37oC in a humidified incubator under 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native chromatin was prepared by lysing CD4+ cells in low salt buffer and digesting with Micrococcal nuclease. Lysates were then sonicated to assist in recovery of oligonucleosomes and adjusted to RIPA immunoprecipitation buffer conditions. Extracts were then incubated overnight with specific antisera prior to immunoprecipitation with protein A & G beads, and recovery of bound DNA using silica column purification.
Twenty five microliters of immunoprecipitated or input DNA (ranging from <1 ng – 4 ng DNA) was used for library preparation using Illumina (San Diego, CA) TruSeq™ DNA Sample Preparation reagents, with ligation of 1/10th the manufacturers recommended adapter amounts and agarose gel selection of DNA fragments in the 200-500 bp size range.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
H3K27me3 ChIP, low cell number, replicate 3
|
| Data processing |
Illumina GAIIx image analysis and base calling was performed using Illumina’s RTA software version 1.4 and experiments performed on the Illumina HiSeq 2000 were analysed using RTA version 1.12.
Reads were filtered to remove those with low base call quality using Illumina’s default chastity criteria.
Reads were mapped to the human reference genome using BWA version 0.5.9 with default settings
Filtering of alignment files to identify uniquely mapping, non-duplicate reads, was performed using samtools version 0.1.15.
Manipulation of alignments for display was performed using bedtools version 2.11.2 software, with visualisation in the IGV genome browser.
Peak calling was performed by MACS version 1.4 [25] using shift size determined by the size of sequencing library inserts, and switching off local background estimation.
Genome_build: hg18
Supplementary_files_format_and_content: bed files of peaks called in experiments scaling down ChIP with H3K4me3 in a benchmark technique, and in new technique from 2 x 10e7 cells / IP to 2 x 10e4 cells / IP
|
| |
|
| Submission date |
Nov 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregor Duncan Gilfillan |
| E-mail(s) |
gregor.gilfillan@medisin.uio.no
|
| Phone |
+47 23016419
|
| Organization name |
Oslo Universitetssykehus
|
| Department |
Medical Genetics
|
| Lab |
Norwegian Sequencing Centre
|
| Street address |
Medisinsk Genetikk
|
| City |
Oslo |
| ZIP/Postal code |
0407 |
| Country |
Norway |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE42147 |
Limitations and possibilities of low cell number ChIP-seq |
|
| Relations |
| SRA |
SRX203345 |
| BioSample |
SAMN01804567 |
