5prime phase lock gel heavy tubes

For RNA extraction of the normotensive pulmonary artery, the adventitial layer was sectioned into 6 pieces and submerged in TRIzol reagent (Invitrogen #15596026, Thermo Fisher Scientific, Waltham, MA, USA). Tissue was homogenized using a BeadBug homogenizer with 1.5 mm zirconium beads. (Benchmark Scientific, Sayreville, NJ, USA). For PAAF experiments, RNA isolation was carried out using TRIzol and 5PRIME Phase Lock Gel Heavy tubes (#2302830, Quantabio, Beverly, MA, USA) and RNA was extracted using either a standard TRIzol total RNA isolation protocol or the RNeasy^® Mini kit (#74104, Qiagen^®, Hilden, Germany), which was then reverse transcribed into cDNA using the NEB cDNA ProtoScript First Strand Kit (#E6300L, New England Biolabs, Ipswich, MA, USA) and then RNA was extracted using the RNeasy Mini kit (#74104, Qiagen, Hilden, Germany). Quantitative real-time PCR was performed using the StepOnePlus^TM Real-time PCR machine (Thermo Fisher Scientific, Waltham, MA, USA) and KAPA SYBR Fast Universal qPCR kit (#KK4601, Roche, Basel, Switzerland) using primers targeting genes of interest listed in Supplementary Table S1 (produced by Integrated DNA Technologies, San Diego, CA, USA). Relative gene expressions were compared against housekeeping gene 18S ribosomal RNA unless otherwise noted.

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).

Cells were harvested in PBS and pelleted. Pellets were resuspended in buffer A [5 mM Pipes (pH 8.0), 85 mM KCl, and 0.5% NP-40 substitute] and incubated on ice for 10 min. After centrifugation (5000 rpm for 5 min at 4°C), the supernatants containing the cytoplasm were removed and the nuclei-containing pellets were washed in buffer A without detergent. Nuclear RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions, using 5PRIME Phase Lock Gel heavy tubes (Quantabio) to separate the aqueous phase from the interphase/organic phase.
Sequencing libraries were generated using the RiboCop rRNA depletion kit followed by the CORALL Total RNA-Seq library preparation kit (both from Lexogen), following the manufacturer’s instructions and thereafter sequenced paired-end 50 bp with a NovaSeq 6000 (Illumina) at the Science for Life Laboratory National Genomics Infrastructure (Stockholm, Sweden).