GSM9682835: sub-tiling of mammalian CRE BC library version 1; Mus musculus; OTHER
Libraries were electroporated in NEB 3020K electro-competent cells. Plasmids from cells were bottlenecked and extracted using the Zymo MidiPrep kits per manufacture's instructions. PCR amplification was performed from the plasmid libraries to generate the CRE-BC libraries. The reaction consisted of 12.5 µl KAPA2G Robust master mix, 1.25 μl 10 μM oJBL060 (appending Nextera P7 adapter), 1.25 μl 10 μM custom-indexed P5 primers (NextP5_index1-9), 0.25 μl of SYBr green 100×, and 8.75 μl water. PCR step was amplified with tracking by qPCR with 1 min at 95 °C, and cycles up to the qPCR inflection point with 15 s at 95 °C, 15 s at 65 °C and 1 min at 72 °C. The resulting amplified libraries are purified by 1.0× Ampure XP beads (Beckman Coulter).  Sequencing libraries were pooled and paired-end sequenced on the NextSeq2000 with the following primers and cycle numbers: read1 (CRE forward): 159 cycles, primer oJBL681/oJBL684; index1 (BC read): 15 cycles, primer oJBL061; read2 (CRE reverse): 158 cycles, primer oJBL683/oJBL686; index2 (sample index): 6 cycles, primer oJBL064. For libraries with larger amplicons, cycle numbers were adjusted: read1: 401 cycles; index1: 15 cycles; read2: 212 cycles; index2: 10 cycles.