GSM6820871: KPK2_t0_rep3; Mus musculus; OTHER
Genomic DNA from collected cell pellets were prepared with purelink mini kit (ThermoFisher) according to the manufacturer's suggested protocol. Libraries were generated by amplification from genomic DNA using a modified 2-step PCR   version of the protocol published by Doench et al. Briefly, we perform an initial “enrichment” PCR, whereby the integrated sgRNA cassettes are amplified from gDNA, followed by a second PCR to append Illumina sequencing adapters on the 5'- and 3'-ends of the amplicon, as well as a random nucleotide stagger and unique demultiplexing barcode on the 5' end. Each “PCR1” reaction contains 25µL of Q5 High-Fidelity 2X Master Mix (NEB), 2.5µL of Nuc PCR#1 Fwd Primer (10µM), 2.5µL of Nuc PCR#1 Rev Primer (10µM), and 5µg of gDNA in 20µL of water (for a total volume of 50µL per reaction). The number of PCR1 reactions is scaled accordingly; therefore, we performed six PCR1 reactions per technical replicate, per time point (T0 or TF), and per condition. PCR1 amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) and used as template for “PCR2” reactions. Each PCR2 reaction contains 25µL of Q5 High-Fidelity 2X Master Mix (NEB), 2.5µL of a unique Nuc PCR#2 Fwd Primer (10µM), 2.5µL of Nuc PCR#2 Rev Primer (10µM), and 300ng of PCR1 product in 20µL of water (for a total volume of 50µL per reaction). We performed two PCR2 reactions per PCR1 product.   PCR Program for PCR1 and PCR2: 1) 98°C x 30s; 2) 98°C x 10s; 3) 65°C x 30s; 4) 72°C x 30s; 5) Go to step 2 x 24 cycles; 6) 72°C x 2 min; 7) 4°C forever. Library amplicons were size-selected on a 2.5% agarose gel, purified using the QIAquick Gel Extraction Kit (Qiagen), and sequenced on an Illumina NextSeq500 instrument (75nt single end reads). Files resulting from 4 sequencing lanes were concatenated into a single file for each condition.