GSM9574339: PGK-Lockwood23; unidentified plasmid; OTHER
Plasmid DNA was extracted from bacterial colonies via Qiagen Maxiprep Sequencing library preparations (Fig4C, Fig6D,E) were performed by setting up 8 20uL PCR reactions (PCR1) using the NEB Next Ultra II Q5 Mastermix (NEB, catalogue number M0544X). 20 ng of plasmid library was used per reaction. Samples were run as a qPCR for approximately 10 cycles. Forward primer mix for PCR1 was an equimolar mix of 8 primers with carrying numbers of degenerate bases (S1-8), sequences are outlined in Supplementary Table 1. PCR1 products were pooled and purified using homemade SPRI beads at at 1X bead ratio, then run on a 2% E-Gel™ EX Agarose Gel (Invitrogen, catalogue number G401002) before being gel extracted. The entire gel extracted product was used as a template for 8 20uL PCR reactions for barcoding and Illumina adapter addition (PCR2). Samples were run as a qPCR for approximately 6 cycles before being pooled and cleaned up using homemade SPRI beads at a 1X ratio. Equal amounts of Old Library and New Library PCR2 products were pooled to make the final sequencing library (Fig6). The sequencing library was diluted to 4nM and denatured with 0.2N NaOH and diluted to 20pM. Sample was loaded at 17pM with ~10% PhiX spike in. Illumina MiSeq was run using the Micro kit in a 1x150bp format. gRNA counts were generated using a custom python pipeline (see Data Availability).