title: GSM4176056: END_seq_PB_WT_REP1; Mus musculus; OTHER
library_construction_protocol: Single cell suspensions of pre-B cells (30 million per agarose plug), primary mouse bone marrow pre-B cells (10 million per agarose plug), and thymocytes (70 million per agarose plugs) were washed in PBS, embedded in agarose and transferred into plug molds (Bio-Rad CHEF Mammalian Genomic DNA plug kit) and processed as previously described (Canela et al., 2016).  Primary mouse bone marrow pre-B cells for END-Seq analyses were obtained from 6-12 week old Vavp-Bcl2-transgenic mice (Ortega-Molina et al., 2015) and cultured in the presence of recombinant IL-7 (ThermoFisher) for 7-10 days at 2 x 106 cells/ml in vitro. To induce cell cycle arrest, cells were resuspended in media without IL-7 and maintained at 2 × 106 cells/ml for 48 hours and harvested for END-seq. END-seq was performed as described in Canela et al., 2017. Agarose plugs were made using the Bio-Rad CHEF Mammalian Genomic DNA plug kit. Embedded cells were lysed and digested using Proteinase K (50º C, 1 hour then  37ºC for 7 hours). Plugs were rinsed in TE buffer and treated with RNase A at 37ºC, 1 hour. The next enzymatic reactions were performed with the DNA in agarose plugs to prevent shearing. DNA ends were blunted for 1 hour at 25ºC with Exonuclease T (NEB). After blunting, A-tailing was performed, followed by ligation of “END-seq hairpin adaptor 1”, 5′-Phos-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[Biotin-dT]U[Biotin-dT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase.  Agarose plugs were then melted and dialyzed and DNA was sonicated to a median shear length of 170bp using Covaris S220 sonicator. DNA was ethanol-precipitated and dissolved in 70 ul TE buffer. Biotinylated DNA was isolated using MyOne Streptavidin C1 Beads (ThermoFisher #650-01), followed by end repair and dA-tailing. The second end was ligated to “END-seq hairpin adaptor 2”, 5′-Phos-GATCGGAAGAGCACACGTCUUUUUUUUAGACGTGTGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Hairpins were digested using USER (NEB), and the resulting DNA fragments were PCR amplified using TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond).  PCR fragments were isolated by size selection from agarose gel, selecting 200-500 bp fragments followed by DNA purification using QIAquick Gel Extraction Kit. Libraries were quantified using KAPA Library Quantification Kit and sequenced using Illumina NextSeq 500 or 550.
identifiers.namespace: GEO
identifiers.id: GSM4176056