title: GSM1861004: SCL-seq naive B cells from lymph node; Homo sapiens; OTHER
library_construction_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.
attributes.tag: GEO Accession
attributes.value: GSM1861004