GSM3728739: H358_PD-L1_Low_LibraryA_Replicate1; Homo sapiens; OTHER
DNA was extracted from the unsorted cells using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer's instructions. Extractions were performed on 4x10^7 cells using 5x10^6 cells per column to ensure enough gDNA for 300X coverage of the library. DNA was eluted by adding 125 µL of water to each column. The same eluate was added back to the column for a second elution. The DNA concentration in the final eluate was assessed using the Qubit dsDNA BR assay kit (Thermo Fisher). To facilitate maximum recovery of gDNA from the sorted cells, a previously described method (Largaespada, et al., 2008) was used with the following modifications: Sorted cell pellets were resuspended in 500 µL of tissue lysis buffer, consisting of 460 µL of STE buffer [1 mM EDTA (pH 8.0), 10 mM Tris-HCl (pH 8.0), 100 mM NaCl] supplemented with 10 µL of 0.5 M EDTA, 10 µL of proteinase K [10 mg/mL in TE buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA], and 20 µL of 10% SDS. Pellets were digested overnight at 55°C while shaking at 1000 rpm on a Thermomixer (Eppendorf). The following day, 5 µL of 2 mg/mL RNase A was added to each tube and incubated at 37°C for 1 hr while shaking at 1000 rpm. Extractions were performed with an equal volume of pH 7.9-buffer saturated phenol, followed by phenol:chloroform:isoamyl alcohol (25:24:1), followed by chloroform. 20 µg of glycogen (Roche) and 1.5 mL of 100% ethanol was added to each tube and DNA was precipitated at -80°C for 1 hr followed by centrifugation at 18,000 g for 10 min at 4°C. Pellets were washed with 1 mL of 75% ethanol, dried, and resuspended in 21 µL of water by incubating at 37°C for a minimum of 4 hrs. DNA concentration was determined with the Qubit dsDNA BR assay kit.  Methods to prepare PCR amplicon libraries for deep sequencing were adapted from a previously published protocol (Shalem, et al., 2014). All primer sequences are provided in Supplementary Table 8. For unsorted cells, an initial round of PCR (PCR I) was performed using 6.6 µg of gDNA per 100 µL PCR reaction. To maintain 300X coverage, 20 reactions were assembled for each sample. For sorted cells, all extracted gDNA for a given sample was distributed into two 100 µL reactions. In both cases, 18 cycles of amplification were performed using Herculase II Fusion polymerase (Agilent). All reactions for a given sample from PCR I were then pooled together and a second round of PCR (PCR II) was performed to add the necessary adapters for Illumina sequencing. Due to variable PCR efficiency between samples, the cycle number for PCR II was adjusted so that each library was amplified in a 50 µL reaction to a common endpoint with respect to DNA quantity (approximately 50 ng of DNA library in a 50 µL PCR sample). DNA was purified for sequencing using AMPure XP beads (Agencourt) according to the manufacturer's instructions with the following modifications: Each 50 uL PCR II reaction was mixed with 25 uL of beads and incubated for 5 minutes. Magnetic separation was used to collect the supernatant. The supernatant was mixed with 90 µL of beads and incubated for 5 minutes. The supernatant was collected and discarded. Beads were washed twice with 200 µL of 70% ethanol and then dried for approximately 12 minutes. Bound DNA was eluted from the beads using 40 µL of water. Constructed as per Shalem et al, Science 2014 and Golden et al, Nature 2017 Genome-scale CRISPR-Cas9 Screen (GeCKO)