Nested-PCR homo_sapiens:sgRNA specific PBS treated pool #2
Genomic DNA from cell pellets containing 1.4ĘxĘ107 cells were extracted using the DNAeasy Blood and Tissue Kit (Qiagen) according to manufacturerŐs protocol. For amplification of the sgRNA containing regions, a total of 25ĘxĘPCR reactions were performed using 1Ęľg genomic DNA per reaction as input Q5 Hot Start HF polymerase (NEB) and primers SEQ-F1 and SEQ-R1 were used for PCR with the following conditions: 98ĘĄC for 2Ęmin, 25Ęcycles of 98ĘĄC for 10Ęs, 62ĘĄC for 15Ęs and 72ĘĄC for 30Ęs, final extension with 72ĘĄC for 2Ęmin. The PCR product was cleaned using QIAquick PCR purification Kit and eluted into nuclease free water. DNA concentration of the eluate was determined using Qubit HS DNA Assay. 5Ęng of the purified PCR product was used for enrichment PCR with Q5 Hot Start HF polymerase (NEB), primers SEQ-F2 and SEQ-R2 and the following PCR conditions: 98ĘĄC for 2Ęmin, 15 cycles of 98ĘĄC for 10Ęs, 72ĘĄC for 15Ęs and 72ĘĄC for 30Ęs, final extension with 72ĘĄC for 2Ęmin. The PCR product was purified with Agencourt AMPure XP beads at a product to beads ratio of 1:1.2. The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100 bp single end sequencing and addition of 20Ę% PhiX Control v3 (Illumina) at a concentration of 8 pM. Two MiSeq runs were performed each containing one replicate of the TRAIL and PBS treated condition.