title: GSM7832006: R1_6; Mus musculus; OTHER
library_construction_protocol: genomic DNA was extracting using QIAGEN Blood & Cell Culture DNA Midi Kit according to manufacturer's instructions. The sequencing library preparation method is a two-step PCR protocol. The first round of PCR amplifies the region of 250-300 bp containing the sgRNA from the genomic DNA with custom primers. The PCR1 forward primer has only the part needed to bind to the flow cell (P5) and vector-specific sequence upstream of the sgRNA insert (about 40nt in length). The primer lacks the Illumina sequencing primer binding site. The PCR1 reverse primer contains the partial adaptor sequence of the NEBNext Multiplex Oligo for single indexing for Illumina (the 3' region after the index) plus vector-specific sequence downstream of the sgRNA insert (about 25 nt in length). The second round of PCR adds at 3' unique single indexes to each library. A 5ul aliquot of PCR1 is taken to perform the PCR2. The PCR2 forward primer is the P5 sequence and the PCR2 reverse primer is a NEBNext Multiplex Oligo for single indexing for Illumina. PCR2 amplicons are purified with magnetic beads. Libraries are sequenced as a pool with a custom sequencing primer to increase complexity in the first 20 nucleotides of the libraries. The sequencing primer are the 44 nt of the vector sequence immediatly upstream of the first nucleotide of the sgRNA where the sequence read begins.