GSM8580433: SVS_DAY14_NT__biol rep2; Homo sapiens; OTHER
The extraction of the genomic DNA (gDNA). Briefly, the gDNA was harvested from each of the collected sample using the Zymo Research Quick-gDNA MidiPrep (Zymo Research) according to the manufacture's protocol. To amplify the sgRNAs for NGS, a PCR reaction was set up for 23 cycles in 96  PCR plates (Sarstedt). Each gDNA sample was processed by using a mix of 8 P5 primers in combination with 1 unique P7 primer. For the purification of the amplified product, the PCR reaction was mixed with 5 volumes of DNA Binding Buffer (Zymo Research), transferred in the Zymo-Spin V column with Reservoir (Zymo Research) and centrifuged at 500 RCF for 5 minutes at room temperature. The column was washed twice with 2 ml DNA Wash Buffer (Zymo Research) by a second step of centrifugation. The column was transferred to a 2 ml collection tube at spined again at maximum speed for 1 minutes to remove residual wash buffer and finally the purified PCR reaction was eluted with Nuclease-Free Water (Invitrogen). The PCR product was quantified from agarose gel.