GSM8859897: AF-NoCT-siPARP12, Bio rep4; Mus musculus; RNA-Seq
MIN6 cells were a gift from the Yamagata lab and were grown in DMEM containing 4.5 g/L each of D-glucose and L-glutamine, 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 50 mM 2-mercaptoethanol maintained at 37 ºC in a 5% CO2 atmosphere. For PARP12 knockdown experiments, cells were treated at 80% confluency using Lipofectamine RNAiMAX (Invitrogen, Cat# 13778150) with SMARTpool ON-TARGETplus non-targeting siRNA (Dharmacon, cat#D-001810-10-20) or siRNA targeting Parp12 (Dharmacon, cat# L-065127-01-0020), with concomitant treatment with CT2 (100 ng/mL IFN-γ: R&D, cat#485-MI-100, 10 ng/mL TNF-α: R&D, Cat#410-MT-010, and 5 ng/mL IL-1β: R&D, cat #401-ML-005) for 24 h. MIN6 cells were harvested using Tri reagent (Zymo Research, R2050-1-200) and the RNA was extracted using RNA Clean & Concentrator™-5 kit (Zymo Research, R1014), following manufacturer instructions. To test the specificity of the ADP-ribosylation-binding AF1521 macrodomain, an RNA aliquot was pre-treated with 2 nM SARS-CoV2 macrodomain MAC1 at room temp for 1 h in hydrolase reaction buffer (250 mM HEPES, 750 mM NaCl, 10 mM β-mercaptoethanol, 0.05 mM Triton X100, 5 mM MgCl2, 1X RNAsecure) to remove ADP-ribosylation. Equal amount of total RNA from each sample was incubated with either 20 µL of Af1521 macrodomain conjugated to magnetic beads (Tulip Biolabs, 2426) or control (naked or mutated Af1521) beads in 600 µL RNA pulldown buffer (10 mM Tris-HCl, 0.6 M NaCl, 0.1% NP40, 1X RNA secure) overnight at 4 C. After overnight incubation, the beads were gently washed with RNA pulldown buffer three times, and 50 µL of Tri reagent was added to elute RNA from the beads, followed by RNA Clean & Concentrator™-5 extraction. RNAs eluted post-incubation with AF1521 and Control (mutant AF1521) beads were processed for cDNA synthesis using SMARTer® Universal Low Input RNA Kit (Takara, cat#634938) according to instruction manual, followed by template library prep using NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (NEB, cat#E7805S). Single-read sequencing of the barcoded cDNA libraries with a read length of 150 were performed on NextSeq 550 plateform using NextSeq500/550 High Output v2.5 150 cycles (cat#20024907).