GSM8265717: GBM22 (T0, T1) - DIPG17 (T0, T1) - KNS42 (T0, T1); Homo sapiens; OTHER
Genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue Kit (QIAGEN #69504) according to manufacturer's instructions. Barcoded sequencing libraries were prepared, pooled and analyzed by paired-end sequencing using NextSeq (Illumina) for each of the three DMG cell lines. The TF domain-focused sgRNA library was comprised of ~6 sgRNA per gene (1,486 genes total, including 1,373 TFs, 62 core-essential positive controls and 51 non-targeting negative controls) Transcription factor (TF) domain-focused sgRNA library (gift from Dr. Christopher R. Vakoc to to Dr. Zhiguo Zhang) was amplified using ElectroMAX Stbl4 Competent Cells (Invitrogen #11635018) according to manufacturer's instructions with a representation > 1,000 and used to produce lentivirus as described above. Cas9-expressing cells were spin-infected with lentivirus of the sgRNA library at 1200 rpm at 25°C for 1 hour, with a MOI ≈ 0.3 and representation >1,000. The cells were cultured with a representation > 1,000 at any time point during the screenings. Samples were collected at starting time point (day 3 post infection, T0) and end time point (12 doubling times after starting time point, T1)