RNA analysis

Tissues were homogenized using the TissueLyser II (Qiagen) and RNA extracted using TRIzol Reagent (Ambion). Removal of genomic DNA and synthesis of complementary DNA was performed using iScript™ gDNA Clear cDNA Synthesis Kit (BIO-RAD). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) in a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems) using a ViiA 7 Real-Time PCR System (Applied Biosystems). The ΔΔCt method was used to determine relative gene expression using cyclophilin A (Ppia) as a reference gene. Forward and reverse primer sequences are listed in Supplementary Table 1. All supplementary material and figures are located in a digital research materials repository (46). For RNA sequencing, RNA was extracted from tissues using TRIzol Reagent (Ambion) and Purelink™ RNA Mini Kit (Invitrogen™). RNA integrity was validated by an Agilent bioanalyzer. Ribosomal RNA depletion was performed with RiboErase (HMR) and samples were prepared according to library kit manufacturer’s protocol, then indexed, pooled, and sequenced on a NovaSeq S4 2×150, targeting ~30 million reads per sample. Detailed methods of RNA sequencing analysis are in the Supplemental Methods.