GSM8329964: Cas12a-SLCxSLC_sublib2_week2_rep1; Homo sapiens; OTHER
For the Cas12a-Metal-SLCxSLC, Cas12a- and Cas9-SLCxEnzymes, and Cas12a- and Cas9-SLCxSLC, gDNA was purified from 2x10e7 (QIAamp DNA Mini Kit), 1.7x10e7 (QIAGEN Genomic-tip 100/G) or 2.2x10e8 cells (QIAGEN Genomic-tip 500/G), respectively. gDNA from Cas12a- and Cas9-SLCxSLC screens was digested with restriction enzymes to improve PCR amplification of sgRNA regions. Up to 1.5 mg gDNA was digested with 200 U PacI (Cas12a-SLCxSLC) or 100 U PacI and 100 U XbaI (Cas9-SLCxSLC) (all NEB) for 48 hours. sgRNA sequences from Cas12a library gDNA samples were amplified with barcoded FW and RV primer that introduce Illumina adapter and read1 sequencing primer binding sites. Up to 1.2 mg gDNA was used as template in 12 ml volume (divided in 96 x 125 µl PCRs) in the following program: 98°C for 30 sec.; 5 cycles: 98°C for 10 sec., 61°C for 30 sec, 72°C for 30 sec; 19 cycles: 98°C for 10 sec., 72°C for 1 min.; 72°C for 2 min. For the samples of the metal transporter and SLC family-wide sub-screen 1, Q5 Polymerase (NEB) was used, SLC vs SLC sub-screen 2-4 and SLC vs metabolic enzymes samples were processed with self-purified Pfu-Sso7d polymerase  and Phusion HF buffer (NEB). Primer sequences are shown in Supplementary Table SX. PCR products we double size selected with DNA purification bads and multiplexed for Illumina sequencing. sgRNA sequences from Cas9 library gDNA samples were first amplified with staggered FW and RV primer that contain partial Illumina adapter sequences at the 5' end using the same PCR program as above. Up to 1.2 mg gDNA was used as template (125 µg/125 µl volume PCR) and amplified with self-purified Pfu-Sso7d polymerase. An aliquot of the pooled amplified PCR products was double size selected with DNA purification bads and purified fragments were barcoded in a second PCRs (98°C for 30 sec; 6-8 cycles: 98°C for 10 sec., 61°C for 30 sec, 72°C for 30 sec; 72°C for 2 min). PCR products we double size selected with DNA purification bads and multiplexed for Illumina sequencing.