GSM5494925: K562_library1_3_gRNA amplification; synthetic construct; OTHER
The enriched PCR procedure was performed as described previously(Hill et al., 2018) with the following modifications. A heminested PCR starting from 40 ng of full-length cDNA was used to enrich for gRNA barcodes. Q5 High-Fidelity DNA Polymerase (New England BioLabs) was used for PCR amplification according to the following PCR protocol: (1) 98°C for 45 s, (2) 98°C for 15 s, 65°C for 20 s, then 72°C for 60 s (14-16 cycles). PCRs were cleaned with Zymo DNA Clean & Concentrator-5 columns (Zymo, Orange County, CA, USA) and 15 µl of a 1:2 dilution of the first PCR and 1:2 dilution of the second PCR were carried in each reaction. Then fragments of length 400-500 bp were selected using 8% TBE gel.