title: GSM2055229: SCL-exo technical replicate 2 E14; Mus musculus; OTHER
library_construction_protocol: Cells were scraped in phosphate buffered saline and were pelleted at 100 g before genomic DNA extraction using a DNeasy Blood and Tissue kit (Qiagen, France). Twenty micrograms of DNA in 300 ml of TE buffer (Tris 10 mM, EDTA 0.1 mM, pH 8.0) were sonicated with a Bioruptor (Diagenode, Belgium) to yield 200 to 500 bp fragments. Each glucosylation and biotinylation reaction was run using reagents from the Hydroxymethyl Collector kit (ref. 55013, Active Motif, Belgium) and 500 ng of sonicated DNA. For SCL-seq, 16 reactions were captured on streptavidin-coated magnetic beads (ref. 11205D, Invitrogen). After 5 washes and elution from the beads according to the manufacturer's protocol, the captured DNA was purified, precipitated and pooled for sequencing library preparation using the TruSeq ChIP Sample Prep Kit (Illumina, ref. IP-202-1012). For SCL-exo, after 6 washes in RIPA buffer (50 mM HEPES pH 7.6; 1 mM EDTA; 0.7% Na-Deoxycholate; 1% NP-40; 0.5 M LiCl) and 2 washes in Tris 10 mM pH 8, the DNA-beads complex was processed as previously described [19]: end polishing, ligation of the P7 exo-adapter, nick repair, lambda and RecJf exonuclease digestion, elution, P7 primer extension, ligation of the P5 exo-adapter, PCR amplification, and finally gel-size selection. The exonuclease-digested DNA was eluted from the beads by incubation in 100 ml of Elution Buffer (95% formamide, 10 mM EDTA) at 90 ⁰C for 5 min, followed by DNA precipitation and resuspension in 20 ml of water. The SCL-seq and SCL-exo libraries were quantified using the KAPA library quantification kit for Illumina sequencing platforms (KAPA Biosystems, KK4824) and 40 bp single-end sequenced in a single lane of a HiSeq 2000 (Illumina) following the manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM2055229