title: GSM3638116: Multi-promoter TRIP pool sgRNA3 mutation profiling reads, UID 2; Mus musculus; OTHER
library_construction_protocol: After incubation of Cas9-sgRNA-transfected mES cells, DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen) and prepared for sequencing. We PCR amplified cell line DNA IR regions of 414bp surrounding the sgRNA target sites using 10µM of PB-cDNA-forward-1-BC primers, containing different index sequences for multiple reactions, and 10µM of PB-cDNA-Reverse-5 primer. The PCR product was sent for Illumina MiSeq. In TRIP pool experiments, we tagged amplicons using 16-bp Unique IDentifiers (UIDs) to be able to detect multiple readings of the same DNA molecules. We performed all Cas9 targeting assays in triplicate. Barcoded IR regions were PCR amplified using Phusion® High-Fidelity DNA Polymerase (NEB). We used Exonuclease-I (20 units) (Enzymatics) for cleavage of single stranded DNA. Purified sequences were sent for Illumina HiSeq. Replicates were assessed for consistency of IR mutation frequencies, and merged for subsequent analyses. TRIP library construction and transfection were performed according to the TRIP protocol (Akhtar et al, Cell 2013; Akhtar et al, Nature Protocols 2014).
attributes.tag: GEO Accession
attributes.value: GSM3638116