GSM6266291: Ascl1 Ngn2 CRISPR-UMI Screen; Mus musculus; OTHER
Genomic DNA isolation was extracted using Phenol-Chlorophorm followed by Isopropanol precipitation. Chromatin was fragmented and sgRNA fragments were excised by PacI digestion (NEB). Size selection was performed using Speed Beads (GE45152105050250; Sigma-Aldrich) . sgRNA sequences were PCR amplified with AATGATACGGCGACCACCGAGATCTACAC-NNNNNN-CGAGGGCCTATTTCCCATGATTCCTTC, where the 6-bp NNNNNN sequence represents specific experimental indices used for demultiplexing samples after NGS, and CAAGCAGAAGACGGCATACGAGATACCGTTGATGAGTAG. NGS was performed using custom primer (CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG) instead of standard illumina primer. For more detail see Michlits et. al. 2017. Read 1: custom primer (first 20bp are sgRNA Sequence) index read1: UMI (first 10bp random nucleotides used as unique molecular identifiers) index read2: 6bp sequences for demultiplexing (see Michlits et al., 2017)