Qiaamp dna columns

The gRNA sequencing and analysis have been previously described (6 (link)). Briefly, genomic DNA from the cells was isolated using QIAamp DNA columns (Qiagen, Hilden, Germany), and gRNA sequences were amplified and barcoded for next-generation sequencing using two-step polymerase chain reaction (PCR). All PCRs were performed using Phusion Flash High-Fidelity Master Mix (Thermo Fisher Scientific, F548L). Sequencing was performed with Illumina HiSeq and 75–base pair single-end reads at the Yale Center for Genome Analysis. Reads were aligned to index sequences using the Bowtie aligner, and a maximum of one mismatch was allowed in the 20–base pair gRNA sequence. The number of uniquely aligned reads for each library sequence was calculated after alignment for each of three biologically independent replicates.

Genomic DNA isolation from both sorted and unsorted cell pellets was performed using QIAamp DNA columns (Qiagen, Hilden, Germany), and gRNA sequences were amplified in a two-step polymerase chain reaction (PCR): For the first PCR, the amount of input genomic DNA for each sample was calculated to achieve 160× coverage of the hGeCKOa and hGeCKOb libraries; a second PCR was performed to attach Illumina adapters and barcodes for next-generation sequencing. All PCRs were performed using Phusion Flash High Fidelity Master Mix (Thermo Fisher Scientific, F548L). Sequencing was performed with Illumina HiSeq and 75–base pair (bp) single-end reads at the Yale Center for Genome Analysis. Primers and barcode sequences are listed in table S5. Reads were aligned to index sequences using the Bowtie aligner, and a maximum of one mismatch was allowed in the 20-bp gRNA sequence. The number of uniquely aligned reads for each library sequence was calculated after alignment for each of three biologically independent replicates.