PCR recovery of sgRNA sequences from gDNA

Multiple PCR reactions were prepared to allow amplification of the total harvested gDNA from a 1000x cell coverage for each sample. For the first round of two nested PCRs, the total volume was 100 uL containing 50 ug sheared gDNA, 0.3 uM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 65C – 10 sec, 72C – 20 sec), 1x (68C – 2 min). All first round PCRs were pooled and a fraction was used as template for the second round PCR. The total volume of the second round PCR was 100 uL containing 2 uL pooled first round PCR, 0.5 uM forward (5′-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5′-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)₆ is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 uM each dNTP, 1x Titanium Taq buffer and 1 uL Titanium Taq (Clontech). PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 sec, 55C – 10 sec, 72C – 20 sec), 1x (68C – 2 min). The resulting PCR product (344 bp) was extracted from a 1% agarose gel. For the orthogonal genetic interaction screen, conditions for the first round PCR were slightly modified to: total reaction volume 80 uL containing 20 ug sheared gDNA and the second round PCR product was 887 bp.

Gel extracted bands from the primary CRISPRa screen were submitted for sequencing on an Illumina HiSeq 2500 platform using paired end 50 kits with the custom sequencing primer 5′-GAGACTATAAGTATCCCTTGGAGAACCACCTTGTTGG-3′ for reading the sgRNA sequence and the Truseq Illumina reverse primer to read out 20 nt random barcode sequences used for generation of technical screen replicates (separation of sgRNA reads into three groups with mutually exclusive barcode sequence bins). For orthogonal dual sgRNA library analysis, single end 50 kits were used and read cycles were split, 25 cycles for Read1 with the sequencing primer above (reading the S.pyogenes sgRNA) and 25 read cycles for the ‘Illumina indexing read’ with the custom indexing primer 5′-TTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTG-3′ (reading the S.aureus sgRNA).