GSM7181268: Lib172; Mus musculus; OTHER
DNA was isolated from MCR cells using the DNeasy Blood & Tissue Kit (Qiagen) with RNaseA (Qiagen) pre-treatment. NGS libraries were prepared by a two-step PCR protocol. First, MCR peptide sequences were PCR amplified from 5 mg of genomic DNA using PCR1_MCR_F 5'- TCTTGTGGAAAGGACGAAACACCGGCTGCTGTGGTGGTGCTGATGG-3' and PCR1_MCR_R 5'-TCTACTATTCTTTCCCCTGCACTGTCCGTTGGTGAAGTAGCACTC-3' with NEBNext Ultra II Q5 Master Mix (NEB) in a 50 mL reaction for 15-35 cycles. Cycle counts necessary to reach but not exceed the linear range were determined by quantitative real time PCR (qRT-PCR) with SYBR Green Supermix (Bio-Rad). PCR products were pooled when multiple reactions were performed for the same sample, concentrated using DNA Clean & Concentrator (Zymo), and size selected by gel electrophoresis and extraction (Qiagen). Purified products were diluted and 1 ng of each sample was PCR amplified using unique barcoded sequencing adapters PCR2_Fx 5'- AATGATACGGCGACCACCGAGATCTACAC[barcode]ACACTCTTTCCCTACACGACGCTCTTCCGATCT[stagger]TCTTGTGGAAAGGACGAAACACCG-3' and PCR_Rx 5'- CAAGCAGAAGACGGCATACGAGAT[barcode]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT[stagger]TCTACTATTCTTTCCCCTGCACTGT-3' with Q5 Hot Start High-Fidelity 2X Master Mix (NEB) for 16 cycles. Libraries were purified using AMPure XP Beads (Beckman Coulter), quantified by qRT-PCR using i5_F 5'- AATGATACGGCGACCACCGAGATCTACAC-3' and i7_R 5'- CAAGCAGAAGACGGCATACGAGAT-3', and pooled to equimolar ratios.