title: GSM3576181: MMd21_rep1; Rattus norvegicus; OTHER
library_construction_protocol: Genomic DNA was extracted using QiAMP kit (Qiagen) and PCR was performed in two steps. First, the input genomic DNA was amplified by 30 cycles in order to achieve 300X coverage over the sgRNA library using the primers F1: TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG TAT CTT GTG GAA AGG ACG AAA and R1: GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTT ATT TTA ACT TGC TAT TTC TAG CTC. Then, to attach Illumina adaptors and barcode samples, a second PCR reaction was carried out by 6 cycles using 20ng purified PCR product and the primers F2: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG, and R2:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG.  Amplicons from the second PCR were gel-extracted, quantified, mixed and sequenced using HiSeq 2500 (Illumina). Rat sgRNA library (Toolgen Rat genome RGEN library v1.0) was constructed by Toolgen (www.toolgen.com). Amplified oligos in oligo library (Type:CTTGTGGAAAGGACGAAACACCG-X20-GTTTTAGAGCTAGAAATAGCAAGTTAAA) with ArrayF ( TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG  ) and ArrayR (ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC  ) were inserted into BsmB1-cut pLKO.1 Lentiviral plasmid.