Chloroform isoamyl alcohol mixture

For HTS, ribosomal RNA depleted double-stranded RNA (dsRNA) was used. The dsRNA was extracted from each sample using a previously published CF-11-based method [22 (link)] that was modified. Briefly, 3 g of tissue was homogenized in 8 mL of GHC buffer. About 7.5 mL of homogenate was mixed with one-third volume of chloroform: isoamyl alcohol mixture (24:1, Sigma-Aldrich, Darmstadt, Germany) and centrifuged for 10 min at 4200× g. Seven mL of the clear homogenate was transferred into a 15 mL centrifuge tube, and the volume adjusted to 20% ethanol (ethanol: homogenate ratio 1:4 vol/vol). Then 0.1 g of Whatman^® CF-11 cellulose powder (Sigma-Aldrich, Darmstadt, Germany) was added to the homogenate and the mixture was rotated for 10 min. The mixture was centrifuged at 4200× g for 2 min and supernatant discarded. The CF-11 was washed and centrifuged twice with 5 mL of STE/20% ethanol buffer (20% ethanol (vol/vol), in 10 mM Tris-HCl pH 8.0, 100 mM NaCl and 1 mM EDTA). A final 3 mL of 80% STE buffer (in 20% ethanol) was added to CF-11 and vortexed to resuspend. The CF-slurry was dispensed into a syringe containing 0.2 gm of autoclaved glass wool fibre (SiO₂) and the CF-11 was dried by gently pressing the plunger to remove any remaining buffer. The dsRNA was eluted in 1 mL of preheated (65 °C) 1× STE buffer.

For total RNA isolation, 650 µl of prepared tissue lysate was transferred along with 325 μl Phenol–Chloroform–isoamyl alcohol mixture (77617, Sigma-Aldrich) into 5PRIME Phase Lock Gel Heavy tubes (2302830, Quantabio), followed by vigorous shaking for 15 s and centrifugation at 16,000g for 5 min. Next, 325 µl of Chloroform–isoamyl alcohol mixture (25666, Sigma-Aldrich) was added and the tubes were shaken again for 15 s, followed by 3 min incubation and centrifugation at 16,000g for 5 min. 350 µl of aqueous phase was collected and used to extract total RNA and using AllPrep DNA/RNA 96 Kit (80311, Qiagen). Isolation was performed according to the manufacturer’s instructions with slight modification to remove DNA contamination from the RNA fraction. For this, the RNA fraction was loaded in the RNeasy® 96 Plate and washed with 400 µl RW1 buffer. 80 µl of DNase I (79254, Qiagen) was added to each well and the RNeasy® 96 Plate was incubated for 15 min at room temperature followed by standard protocol starting with washing the 96-well plate with RW1 buffer. Either 500 ng (dCas9-VPR, Ldlr and Pcsk9) or 1 µg (Serpina1(a-e)) of total RNA was reverse-transcribed into copy DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems).

All stock reagents were purchased from commercial suppliers at appropriate stock concentrations, and only SDS and PVP40 were purchased as powders and were weighed and added directly to extraction buffers. The stock solutions UltraPure™ 1 m Tris/HCl (pH 7.5; Thermo Fisher Scientific), sodium chloride solution 5 m (Sigma‐Aldrich), UltraPure™ 0.5 m ethylenediaminetetraacetic acid (EDTA), (pH 8.0; Thermo Fisher Scientific), sodium dodecyl sulfate (SDS; Sigma‐Aldrich), polyvinyl pyrrolidone (PVP40; Sigma‐Aldrich), 2‐mercaptoethanol (Sigma‐Aldrich), UltraPure™ DNase/RNase‐free distilled water (Thermo Fisher Scientific), acid‐phenol: chloroform (pH 4.5) with isoamyl alcohol at a final ratio of 25 : 24 : 1 (Thermo Fisher Scientific), chloroform/isoamyl alcohol mixture (Sigma‐Aldrich), lithium chloride (8 m; Sigma‐Aldrich), ethanol absolute for analysis (Merck), chloroform for analysis (Merck), and RNaseZap™ RNase decontamination wipes (Invitrogen) were of molecular biology grade and were free of RNAses, DNAses, and pyrogens. All plastic supplies for the preparation of extraction buffer and the tubes used for extraction were disposable and were free of RNAses, DNAses, and pyrogens. We avoided the use of reagents with acute toxicity, such as diethyl pyrocarbonate, which is frequently used to inactivate RNAses.