title: GSM3756328: CAST pure rep 2; Mus musculus; OTHER
library_construction_protocol: DNA was extracted from cells using DNeasy-kit (QIAGEN) and eluted twice with 2 × 200 µl of the kit elution buffer (AE), concentrated using 1.8x Agencourt AMPure XP beads (Beckman Coulter) and eluted in 50 µl EB (Qiagen). DNA concentrations were measured using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).  DNA samples were sonicated   in the M220 Focused-ultrasonicator (Covaris) using 50 W, 20% Duty Factor at 20°C for 120 s, in order to reach an average target peak size of 250 bp as verified with the D1000 or D1000 High Sensitivity ScreenTape using the Electrophoresis 2200 TapeStation system (Agilent). Library preparation was carried out as described [Yehuda, 2018 #6334]. Briefly, sonicated DNA was subjected to a 50 µl end repair reaction using 1 µl End repair mix (E6050L, NEB), cleaned by 1.8× Ampure XP beads, followed by a 50 µl A-tail reaction using 2 µl Klenow fragment exo- (M0212L, NEB). The products were cleaned by 1.8× beads and ligated by 2 µl quick ligase (M2200, NEB) to 0.75 µM Illumina compatible forked indexed adapters. Ligation products were size selected by 0.7× PEG (considering the PEG in the ligation buffer) in order to remove free adaptors. 12–19 cycles of amplification were performed by PFU Ultra II Fusion DNA polymerase (600670, Agilent) with the following Primers:   P7: 5' AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGAC 3'; P5: 5' CAAGCAGAAGACGGCATACGAGAT 3'. Amplified DNA was size selected for 300–700 bp fragments by taking the supernatant after using 0.5× beads (which removed fragments greater than 700 bp) followed by a 1.0× cleaning to remove remaining primers and adapter dimers. The final quality of the library was assessed by Qubit and TapeStation. Libraries were pooled and sequenced on NextSeq (Illumina) by 75 bp paired-end sequencing, generating 10 M reads per library.