|
| Status |
Public on Oct 30, 2015 |
| Title |
Input DNA from BN358 P1 cells |
| Sample type |
SRA |
| |
|
| Source name |
in vitro differentiated plasmablasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: plasmablasts
origin: differentiation of NBC from buffy coat (healthy donor)
|
| Growth protocol |
Naive B cells from healthy donors were purified by negative selection using magnetic cell separation (Miltenyi Biotech) and cultured at 7.5.105 cells/ml with the stimulation cocktail 1 (2,6 µg/ml Fab'2 anti-Ig, 100 ng/ml CD40 Ligand, 1 µg/ml CpG ODN 2006 and 50 U/ml IL2) for 4 days. Activated B cells were then washed and further cultured at 4.105 cells/ml with the differentiation cocktail 2 (50 U/ml IL2, 5 ng/ml IL4, 10 ng/ml IL10)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the AllPrep DNA/RNA mini kit (Qiagen) after cell-sorting (FACS Aria, BD) of blood or lymph node naive B cells and of in vitro differentiated plasmablasts (day 6 of the culture). For ChIP experiments, cells were fixed with 1% formaldehyde prior to cell sorting.
Genomic DNA (7 μg) from naïve B cells and plasmablasts was fragmented to 200 to 500 bp by sonication (Bioruptor, Diagenode) before incubation with β-glucosyltransferase and azide-glucose. Glucosylated 5hmCs were then labeled with biotin before enrichment of the biotinylated DNA fragments with streptavidin-coated magnetic beads. All steps used reagents from the Hydroxymethyl Collector kit (Active Motif). After elution from the beads, purified DNA was precipitated and processed for sequencing on a Highseq2000 (Illumina). Library preparations and sequencing reactions were run at the IGBMC genomic platform (Strasbourg, France). ChIP-seq was performed in P1 cells as follows. Cells were fixed for 8 minutes in 1% formaldehyde at room temperature and chromatin was sonicated for 15 minutes with the Bioruptor Sonication System (Diagenode). Immunoprecipitation was carried out with antibodies from Diagenode against H3K4me1 (pAb-194-050, lot: A1863-001P) and H3K27ac (pAb-196-050, lot: A1723-0041D) using approximately 500,000 cells per antibody. ChIP-seq libraries were constructed using the Kapa Hyper Prep Kit (Kapa Biosystems) and sequenced with an Illumina HiSeq1500 sequencer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
SCL-seq fastq files were mapped to human hg19 genome assembly using Bowtie with parameters l=32 bp, n=1, m=1, strata, best, and Samtools.
The bam files were then processed to generate .wig files using MACS 1.4.0.
Genome_build: hg19
Supplementary_files_format_and_content: wig files
|
| |
|
| Submission date |
Aug 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Gilles Salbert |
| E-mail(s) |
gilles.salbert@univ-rennes1.fr
|
| Phone |
+33 223 236 625
|
| Organization name |
University of Rennes 1
|
| Department |
Biology
|
| Lab |
UMR6290 CNRS
|
| Street address |
Avenue du Général Leclerc
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL18460 |
| Series (2) |
| GSE72360 |
Mapping of 5-hydroxymethylcytosine by selective chemical labeling (SCL-seq) in human naive B cells and in vitro differentiated plasmablasts (P1 cells) [HTS] |
| GSE72498 |
Cell cycle-dependent reconfiguration of the DNA (hydroxy) methylome during terminal differentiation of human B cells into plasma cells |
|
| Relations |
| BioSample |
SAMN04011594 |
| SRA |
SRX1164305 |
