Project: mRNA-seq on PLSCR1 KO Huh-7.5 cells; Cells: Huh-7.5-Cas9 PLSCR1 KO; Analysed data: See Manuscript Supp Tables 14 and 15
Cell culture and CRISPR KO: 30,000 Huh-7.5 cells were seeded in five wells of a 24-well plate with 480 uL media. The cells were reverse transfected with 120 uL of a transfection mixture composed of 250 nM of pooled anti-PLSCR1 or non-targeting Edit-R crRNAs from Horizon Discovery (cat. CM-003729-01-0002, CM-003729-02-0002, CM-003729-03-0002, and CM-003729-04-0002 or U-007501-01-05, U-007502-01-05, U-007503-01-05, and U-007504-01-05, respectively) which had been resuspended with an equimolar amount of Edit-R tracrRNA (Horizon, cat. U-002005-20) and a 1:200 dilution of Dharmafect 4 (Horizon, cat. T-2004-01). The following day, the media was changed, and the cells were progressively scaled up to a 6-well plate over the next 4 days. When the cells were confluent in the 6-well plate, the media was removed from four of the wells. They were then washed with 1x PBS (cat. 14190-144) and lysed with 1 mL TRIzol (Life Technologies, cat. 15596-018) for 5 minutes at room temperature before transferring to an Eppendorf tube and freezing at -80C to await RNA extraction. The remaining well was lysed with 300 L of RIPA buffer (Thermo cat. 89900) supplemented with 1x protease inhibitor (Thermo cat. 87786) and 1x EDTA and prepared for western blot as described below, in the Western Blots section. RNA extraction: 2 ml MaXtract High Density tubes (Qiagen, 129056) were centrifugated at 12,00016,000 x g for 20-30 second centrifugation. A volume of 750 L TRIzol-prepared sample was combined with 150 L chloroform in these tubes and hand-shaken vigorously. Phase separation was accomplished by centrifugation at 1500 x g for 5 min at 4C. The aqueous phase was then mixed with 400 L ethanol 95-100% in a separate tube. These preparations were then transferred to Zymo Research RNA clean and concentrator-25 kit columns (Zymo Research, cat. R1018) and subjected to multiple wash and centrifugation steps as recommended by the manufacturer. An in-column DNase I treatment was performed using Qiagen DNase (Qiagen, 79254). Finally, RNA was eluted with 50 L DNase/RNase-Free water and stored at -80C. Sequencing: Poly A-enriched libraries were made using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, cat. E7770) and sequenced on a NovaSeq SP with PE150 read length.