title: GSM8527590: WT_week7_rep1; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was purified using QIAamp DNA Mini columns (Qiagen) gDNA was digested overnight with 20U PacI (NEB) and double size-selected to enrich the sgRNA containing fragments (380 bp). Briefly, 0.7 x vol AmpliClean magnetic beads (Nimagen) were added to the sample to bind large gDNA fragments; the cleared supernatant was added to fresh beads (0.6 x volume of the original gDNA sample) to bind the smaller fragments. After size selection, sgRNA sequences were amplified in  with Q5 Polymerase (NEB) using the program recommended by the manufacturer (28 cycles, 65°C annealing temperature). PCR products were further double size-selected to remove residual large gDNA fragments as described above. Purified PCR products were used in barcoding PCRs to add Illumina adapters and indices for pooled sequencing. A detailed protocol can be found on Addgene (https://www.addgene.org/pooled-library/human-transporters/).