|
| Status |
Public on Nov 24, 2015 |
| Title |
Promoter Capture Hi-C, Jurkat T lymphocyte cells, replicate one & two |
| Sample type |
SRA |
| |
|
| Source name |
Jurkat T lymphocyte cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Jurkat
restriction enzyme: HindIII
|
| Biomaterial provider |
LGC standards; http://www.lgcstandards-atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Human/Alphanumeric/TIB-152.aspx
|
| Treatment protocol |
Cells were fixed in 2% formaldehyde for 10 minutes at room temperature, followed by the addition of cold 1M glycine to a final concentration of 0.125M for 5 minutes at room temperature, then by 15 minutes on ice.
|
| Growth protocol |
Jurkat cells were cultured in RPMI 1640/20 mM L-glutamine supplemented with 10% fetal bovine serum in 25cm2 vented culture flasks at 37C/5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were thawed on ice and re-suspended in 50ml freshly prepared ice-cold lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal CA-630, one protease inhibitor cocktail tablet). Routinely, two pellets from each cell line were re-suspended and combined in 7ml complete lysis buffer to give ~50-60million cells. Cells were lysed on ice for a total of 30 min, with 2x10 strokes of a Dounce homogeniser with a 5 minute break between Douncing. Following lysis, the nuclei were pelleted and washed with 1.25xNEB Buffer 2 then re-suspended in 1.25xNEB Buffer 2 to make aliquots of 5-6million cells for digestion. Following lysis, Hi-C libraries were digested using HindIII then prepared as described in van Berkum et al with modifications described in Dryden et al.
Pre-Capture amplification was performed with 8 cycles of PCR on multiple parallel reactions from Hi-C libraries immobilised on Streptavidin beads which were pooled post-PCR and SPRI bead purified. The final library was re-suspended in 30µl TLE and the quality and quantity assessed by Bioanalyzer and qPCR. Hi-C samples corresponding to 750ng were concentrated in a Speedvac then re-suspended in 3.4μl water. Hybridisation of SureSelect custom promoter and region capture libraries to Hi-C libraries was carried out using Agilent SureSelectXT reagents and protocols. Post-capture amplification was carried out using 6 cycles of PCR from streptavidin beads in multiple parallel reactions, then pooled and purified using SPRI beads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
This Sample represents 2 replicates
|
| Data processing |
Library strategy: Hi-C
Casava software (v1.8, Illumina) was used to make base calls.
reads failing Illumina filters were removed before further analysis
Sequences were output in FASTQ format, poor quality reads truncated or removed as necessary, using Trimmomatic version 0.3023, and subsequently mapped to the human reference genome (GRCh37/hg19) and filtered to remove experimental artefacts using the Hi-C User Pipeline (HiCUP, http://www.bioinformatics.babraham.ac.uk/projects/hicup/).
Off-target di-tags, where neither end mapped to a targeted fragment, were removed from the final data sets using a combination of BEDTools and command line tools.
Genome_build: hg19
Supplementary_files_format_and_content: BEDPE files include on-target read counts
Supplementary_files_format_and_content: BED files include capture bait coordinates (region_baits_coordinates.bed.gz & promoter_baits_coordinates.bed.gz)
|
| |
|
| Submission date |
Jun 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Paul Martin |
| E-mail(s) |
paul.martin-2@manchester.ac.uk
|
| Organization name |
Univeristy of Manchester
|
| Department |
Centre for Genetics and Genomics Versus Arthritis
|
| Street address |
Oxford Road
|
| City |
Manchester |
| State/province |
Greater Manchester |
| ZIP/Postal code |
M13 9PT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE69600 |
Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci |
|
| Relations |
| BioSample |
SAMN03763369 |
| SRA |
SRX1050327 |
