title: Amplicon sequencing data of sgRNA cassettes from pig CRISPR screening time course to identify essential genes in PK15 and IPEC-J2 cell lines
design: Amplicon sequencing data of sgRNA cassettes from pig CRISPR screening time course to identify essential genes in PK15 and IPEC-J2 cell lines
library_name: PK15 Day21_p
library_construction_protocol: Cells from two porcine cell lines (PK15 and IPEC-J2) were transduced with a lentiviral CRISPR library (pGeCKO) at a multiplicity of infection (MOI) of 0.3. Following puromycin selection, cell populations were maintained under standard culture conditions, and samples were harvested at multiple time points post-transduction. For IPEC-J2, samples were collected at days 3, 10, and 26. For PK15, samples were collected at days 5, 13, 21, and 26. At each time point, a minimum of 1×10⁷ cells were harvested per sample for genomic DNA extraction. IPEC-J2 and PK15 porcine cell lines were maintained in DMEM (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific) and penicillin-streptomycin (ThermoFisher Scientific) at 37 °C in a humidified incubator with 5% CO₂. Cells were subcultured at a seeding density of 1.4×10⁴ cells/cm² (IPEC-J2) or 4.6×10⁴ cells/cm² (PK15) every 3–4 days. During GeCKO screening, transduced cell populations were cultured under puromycin selection (2 µg/mL). Genomic DNA was extracted from up to 2×10⁷ cells per sample using the Quick-DNA Midiprep Plus Kit (Zymo Research) according to the manufacturer's instructions. DNA concentration was measured using the Qubit Broad Range Kit (Invitrogen), and quality was assessed using both gel electrophoresis and the Agilent TapeStation with the genomic DNA kit. CRISPR guide RNA regions were PCR amplified from genomic DNA to prepare amplicon sequencing libraries. For each sample, up to 23 µg of DNA was divided across multiple 50 µL PCR reactions using NEBNext Ultra II Q5 master mix (NEB), 0.5 µM of each primer, and up to 1.5 µg DNA per reaction. Custom primers (P5 ARGON and P7 BEAKER) were used, each incorporating Illumina adapter sequences, barcodes (P7), and stagger regions (P5), with phosphorothioated 3' ends. PCR conditions: 98 °C for 3 min; 25 cycles of 98 °C for 10 sec, 56 °C for 10 sec, 72 °C for 25 sec; final extension at 72 °C for 2 min. Reactions were pooled and purified using two rounds of AMPure XP bead cleanup (1:1 bead:sample ratio, 20 µL elution).