title: RNA-seq of human cell line HepG2 edited with CRISPR and treated with splice-switching oligo inhibiting a pseudoexon in PCCA against control SSO and control HepG2 cells
design: RNA-seq of human cell line HepG2 edited with CRISPR and treated with splice-switching oligo inhibiting a pseudoexon in PCCA against control SSO and control HepG2 cells
library_construction_protocol: Cells were washed in PBS and harvested for RNA extraction after 48 hours following SSO transfection. Cells were cultured at 37°C, 5% CO2, in RPMI-1640 medium (Sigma Aldrich, United Kingdom) supplemented with fetal bovine serum (10%), L-glutamine (4 mM), and penicillin (100 U/ml)/streptomycin (100 µg/ml). CRISPR-edited HepG2 cells were transfected with splice-switching antisense oligonucleotides (SSO); RNA oligonucleotides with 2'-O-methyl modifications and phosphorothioate backbones (LGC Biosearch Technologies, Denmark), at 20 nM final concentrations. Cells were transfected with a non-targeting control SSO (5'-AUGUUCUAUGAAAUUCACAGUGUUG-3') or SSO 2 targeting a pseudoexon sequence in PCCA intron 14 (5'-GCUCAAUAUGCUACUGCCAUGCUUG-3'). SSO transfection was performed at the time of cell seeding (200,000 cells/well in 12-well plates; 3.5 cm2/well) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA) and harvested for RNA extraction after 48 hours.  Gene editing was performed using the Alt-R CRISPR-Cas12a (Cpf1) system (Integrated DNA Technologies, IA, USA) for homology-directed repair in HepG2 cells. Cas12a:crRNA (5'-UAAUUUCUACUCUUGUAGAUAGGACAUAAACUGAUGUUCUAUG-3') ribonucleoprotein was co-transfected with a 75 nt single-stranded DNA donor template of the PCCA c.1285-1416A>G sequence variant (¬¬¬-37/+37 nt) using Lipofectamine CRISPRMAX Transfection Reagent (Invitrogen, CA, USA). Cell cultures were expanded from single cell colonies and genotypes were verified by Sanger sequencing: HepG2 PCCA MUT cells functionally hemizygous for PCCA c.1285-1416A>G with a large complex deletion on the other allele (spanning from intron 12 to intron 14), HepG2 PCCA Del7 cells homozygous for PCCA c.1285-1411delTAGAACA, and HepG2 PCCA WT with no editing introduced in the PCCA gene. Total RNA was extracted from cells using TRIzol Reagent (Ambion, CA, USA) by phase separation with chloroform and precipitation in isopropanol. RNA purity was estimated on a NanoDrop 2000c spectrophotometer, and RNA integrity was estimated on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent Technologies, CA, USA). RNA with a 260/280 absorbance ratio >1.8 and RNA integrity number (RIN) >8.0 was used for library preparation.  Library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, MA, USA) with rRNA depletion. Size estimation of the amplified cDNA libraries was performed on an Agilent 2100 Bioanalyzer using the DNA 1000 Kit (Agilent Technologies, CA, USA), and concentrations were estimated by qPCR using the KAPA Library Quantification Kit (Roche, South Africa).