Zymo quick gdna miniprep kit

Vials of banked mononuclear cells from patient bone marrow aspirate were warmed to 37°C and transferred to a sterile 15 mL conical tube. Pre-warmed, 25% fetal bovine serum (FBS) in Iscove’s Modified Dulbecco’s Media (IMDM) was added dropwise to the cells at a rate of approximately 2-3 seconds/ml for 10 mL. Cells were centrifuged at 1,000 rpm for 10 minutes, and the supernatant was gently aspirated. The addition of media, centrifugation, and removal of supernatant was repeated. Cells were then washed once in 1X PBS. gDNA was isolated using the Zymo Quick-gDNA Miniprep Kit (Zymo, Cat # D3025), using the manufacturer’s instructions.

The Genome-Wide CRISPR Knockout (Gecko) v2.0 library containing 120,000 gRNA sequences was used. Lentiviruses were produced by co-transfecting 293T cells and then concentrating 1000 × to increase the viral titer. A total of 1 × 10⁸ C11 cells were infected at a low multiplicity of infection (MOI) (0.1~0.3), to ensure that most cells received only one viral construct with a high probability. Twenty-four hours after transduction, cells were selected with 2 μg/ml puromycin for at least 2 weeks and harvested. The proportion of GFP-positive C11 cells increased after infection with CRISPRV2.0. GFP-positive cells were sorted for 4 rounds and enriched genes. gDNA was extracted using the Zymo Quick-gDNA Miniprep kit (Zymo Research, Orange, CA).
PCR was performed with the following primers: 5′-TCTTTCCCTACACGACGCTCTTCCGATCTTAACTTGAAAGTATTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG -3′ and 5′-CTGGAGTTCCTTGGCACCCGAGAATTCCAATTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′ annealing up- and downstream of the gRNA sequence.