title: GSM8353511: inVivo134; Homo sapiens; OTHER
library_name: GSM8353511
library_construction_protocol: sgRNA-pool containing lentiviruses were transduced into Cas9 expressing KP4 and HPAFII cell lines (in presence of 8μg/ml polybrene), at an estimated MOI = 0.2. After 24h, the lentivirus containing media was removed, cells were washed with PBS, and puromycin-containing media (2μg/ml) was added to the cells for 48-72h until all control cells (not virus-infected) were dead. Timepoint 0 was collected 7 days after the sgRNA transductions. With timepoint 0, 1/3 of cells were stored to -80C as timepoint 0, 1/3 was implanted into pancreas of the athymic mice model and the remaining 1/3 of cells were continued to culture in vitro, as a control for the in vivo screen function. For KP4 approx. 5 million cells were implanted into each mouse and for HPAFII we implanted 2 million cells per mice. Tumor formation was monitored for 29 days, followed by sacrificing the animals and tumor collections. The genomic DNA (gDNA) from the tumors were collected by using DNeasy Blood and tissue kit (Qiagen). NGS library preparations were done in the following way: 10 μg of gDNA, theoretically corresponding to 1.5 million diploid cells, was used as PCR template in 3 parallel PCR1 reactions (3.3 μg template DNA each) using ExTaq DNA polymerase (Takara bio). After 18 cycles, the reactions were pooled. 2 μl of pooled PCR1 product was used as template for PCR2 which was run with qPCR with index primers and stopped before the amplification started to saturate. The resulting products of approx. 360 bp were gel purified (GeneJet), pooled together and Next generation sequenced.