title: GSM7998529: Tbxt_insRCS2_homo_mouse_male; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was purified from mESCs or ear punches of mice of interest using Zymo Quick-DNA Miniprep Plus Kit (Cat. No. D4068) according to the manufacturer's protocol. DNA sequencing libraries compatible for Illumina sequencers were prepared following the standard protocol. Briefly, 1µg of gDNA was sheared to 500-900 base pairs in a 96-well microplate using the Covaris LE220 (450 W, 10% Duty Factor, 200 cycles per burst, and 90-s treatment time), followed by purification with a DNA Clean and Concentrate-5 Kit (Zymo Research, Cat. No. D4013). Sheared and purified DNA were then treated with end repair enzyme mix (T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase, NEB, Cat. No. M0203, M0210 and M0201, respectively), and A-tailed using Klenow 3'-5'exo- enzyme (NEB, Cat. No. M0212). Illumina sequencing library adapters were subsequently ligated to DNA ends, followed by PCR amplification with KAPA 2X Hi-Fi Hotstart Readymix (Roche, Cat. No. KR0370). Following library preparation, custom biotinylated probes were prepared as bait through nick translation, using BAC DNA and/or plasmids as the template. The probes were prepared to comprehensively cover the whole locus. We used BAC lines RP24-88H3 and RP23-159G7, purchased from BACPAC Genomics, to generate bait probes covering mouse Tbxt locus and ~200kb flanking sequences in both upstream and downstream regions. The pooled whole-genome sequencing libraries were hybridized with the biotinylated baits in solution and purified through streptavidin-coated magnetic beads. Following pull-down, DNA sequencing libraries were quantified with Qubit 3.0 Fluorometer (Invitrogen, Cat. No. Q33216) using a dsDNA HS Assay Kit (Invitrogen, Cat. No. Q32851). The sequencing libraries were subsequently sequenced on an Illumina NextSeq 500 sequencer in paired-end mode.