Heavy phase lock gel tube

RNA was isolated from samples collected in TRIzol (Invitrogen) using a chloroform-isopropanol protocol. 200μL chloroform was added to 1mL TRIzol samples, transferred to a heavy phase-lock gel tube (QuantaBio), and spun in a microcentrifuge at 12,000xg for 15 min at 4°C. The aqueous layer was transferred to a new tube containing 1μL of glycogen (Fisher Scientific) and equal volume isopropanol added. After 10 min of incubation at room temperature, sample was spun at 20,000xg for 20 min at 4°C, then RNA pellet was washed with fresh 75% ethanol before drying and resuspending in 40μL nuclease-free water. Isolated mRNA was DNase treated using Turbo DNase (Invitrogen) in reactions consisting of 11 μL (~15μg) RNA and 1.5μL Turbo DNase according to manufacturer’s protocol. DNase-treated RNA was purified using lithium chloride precipitation as described in mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen). 2μg RNA per sample was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen) and MPRA-specific primer (Table S7, primer #2).

For RNA-seq on BMDMs after lung CD4 T cell co-culture (Figs 2 and S2), four independent experiments were performed. For each experiment, BMDMs were seeded in 24-well plates, infected as described, and co-cultured with lung CD4 T cells isolated as described. At 24 hours postinfection, CD4 T cells were separated from BMDMs using CD4 (L3T4) Dynabeads from Invitrogen (11445D) according to the manufacturer’s protocol, and BMDMs were lysed in 1 mL TRIzol (Invitrogen). Total RNA was extracted using chloroform and 2 mL Heavy Phase Lock Gel tubes from QuantaBio (2302830), and the aqueous layer was further purified using RNeasy Mini spin columns from Qiagen (74104). RNA quality was determined using an Agilent 2100 Bioanalyzer and RNA concentration was determined using the Qubit Quantitation Platform at the Functional Genomics Laboratory of The California Institute for Quantitative Biosciences (University of California, Berkeley). RNA-seq libraries were prepared by the DNA Technologies and Expression Analysis Core (University of California, Davis) and differential gene expression was analyzed by the Genome Center and Bioinformatics Core Facility (University of California, Davis). Data displayed as “RNA-seq reads” were normalized to counts per million.