GSM8676228: K562, T0, Replicate 1, 2023 screen; Homo sapiens; OTHER
For the 2023 screen, cell pellets were processed to generate sequencing libraries as follows. Genomic DNA was extracted using the NucleoSpin® Blood XL, Maxi kit for DNA from blood (Macherey-Nagel) with an RNase A treatment step during cell lysis. For the 2022 screen, the extraction was performed as describe above except that the RNase A treatment step was performed separately after extraction of genomic DNA followed by ethanol precipitation. The sgRNA loci were amplified with an indexing 5′ primer (5′-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNCAGCACAAAAGGAAACTCACC) and an indexing 3′ primer (5′-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNGGCGGTAATACGGTTATCCA). Each reaction (100 μL total volume) contained 10 μg of genomic DNA (measured on NanoDrop) and 1 μM of each primer and was run on the Bio-Rad C1000 thermal cycler with the following program: 1 cycle of 30 seconds at 98 °C; 20 cycles of 10 seconds at 98 °C, followed by 75 seconds at 65 °C; 1 cycle of 5 minute at 65 °C; 4 °C hold. At least 400X coverage was maintained for each sample during PCR amplification. Aliquots of the PCR products were purified for sequencing using SPRIselect Reagent (Beckman Coulter) in two double-sided 0.45X-0.8X reactions and two 0.8X reactions. Indexed samples were pooled in equal molar ratio prior to sequencing.