title: GSM8857726: chr1:241498239 T/C variant reporter cells, GFP positive cells, replicate 1; Homo sapiens; OTHER
library_construction_protocol: After sorting, cells were pelleted at 400xg for 4 min at room temperaure and frozen at -20°C. DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen; 51106), following manufacturers protocol and concentrated using Zymo DNA Clean & Concentrator-5 kit (Zymo; D4014), following manufacturers protocol. Samples were eluted 2x with 12 µl of Zymo elution buffer. The first step PCR was performed with up to 250 ng of DNA using 2x Q5 hot start polymerase (NEB M0494), 2.5 µl 10 µM of pooled forward oligos, 2.5 µl 10 µM of reverse oligo that targets sequence on Lenti-CRISPR-V2, and 0.5 µl 25x sybr green (Thermo Fisher Scientific S7563) in 50 µl reactions. If a sample had more than 250 ng of DNA, multiple PCRs per sample were set up to amplify the entirety of DNA from that sample. PCR was run on a light cycler (QuantStudio3 by Thermo Fisher Scientific) with the following parameters 98C for 30s; [98C for 10s; 61C for 20s; 72C for 15s] x 40. Amplification was tracked and PCRs were stoped 2 to 3 cycles after exponential increase. After first step PCR, PCR products were cleaned using 1X Ampure XP beads (Beckman A63881). If multiple PCRs were performed per sample, those PCRs were pooled. The beads were mixed the pooled PCRs, incubated for 10 minutes at room temperature, washed 2 x 30 sec with 75% EtOH, dried for 5 minutes, and resuspended and eluted in 30 µl H2O. Products were quantified by Qubit™ dsDNA HS Assay Kit (Q32854). Second step PCRs were performed on the entirety of product from the first step PCRs, split into multiple PCRs with a maximum of 20 ng template for each. PCRs were performed with a common forward oligo, and specific barcoded reverse oligos (Supplemental Table 4. This second PCR was performed with Q5 hot start polymerase (NEB M0494), 1 µl 10 µM of forward oligo, 1 µl 10 µM of reverse oligo and 20 ng of DNA with the following parameters: 98C for 30s; [98C for 10s; 61C for 20s; 72C for 15s] x 5. The multiple PCRs performed per sample were pooled and cleaned again using 1X Ampure XP beads following the same protocol as above but eluted in 22 µl and were submitted for Tape Station analysis DNA 1000 regular sensitivity. The barcoded PCR products were then pooled into equimolar pools, grouped by sample type, and purified on a non denaturing 10% polyacrylamide gel as previously described 10 (38328795). After gel purification samples were cleaned on Zymo DNA columns and submitted again for Tape Station analysis DNA 1000 regular sensitivity. The final three samples were pooled at equimolar amounts and sequenced with a MiSeq Nano kit.