Direct zol rna microprep kit

Total RNA was purified from the tumor cells using the Direct-zol RNA Microprep Kit, according to the manufacturer’s instructions (Zymo Research, Irvine, CA, USA). RNA sequencing was performed by MedGenome Inc. (Foster City, CA, USA). Reference genome (mm10) and gene model annotation files from Ensembl were used for aligning reads using STARv2.5. The reads were quantified using HTSeqv0.6.1. A differential gene expression analysis was performed using DESeq2 v1.36. Ranked-log2 fold changes from the DESeq2 analysis were used as inputs for the analysis [61 (link)]. GO pathway enrichment was performed with clusterProfiler V4.0. A heatmap was generated using ComplexHeatmap v2.12.0. A ferroptosis pathway map was generated in Pathview v1.36 based on KEGG collections of the mouse ferroptosis pathway gene set. The entire dataset is deposited in GEO (accession # GSE213856).

Sorted cells were resuspended in TRIzol LS (Invitrogen 10296010) and RNA was extracted using the Direct-zol RNA Microprep kit (Zymo Research R2062). cDNA was synthesized and pre-amplified as described previously (Tsinman et al., 2021 (link)). Briefly, isolated RNA was reverse transcribed into cDNA using the SuperScript IV VILO Master Mix with ezDNase Enzyme (Invitrogen 11766050). cDNA was pre-amplified for 15 cycles using the Preamp Master Mix (Fluidigm 100–5580) with a pool of all TaqMan Gene Expression Assays (Applied Biosystems 4351372), except those for the highly expressed genes 18s and Col1a1 (see Supplementary Table S1 for list of all genes and corresponding TaqMan Assay ID numbers).

CUGU and control LNAs were transfected into dissociated DRGs and 24 h later the DRGs were lysed in RIP buffer (150 mM KCl, 25 mM Tris-HCl, 5 mM EDTA, 0.5 mM dithiothreitol, 0.5% NP40,+protease inhibitors). The cleared lysate was incubated with an APC antibody (Santa Cruz Biotechnology, sc-896; 1:500) overnight at 4 °C. Antibody–protein–RNA complexes were precipitate by incubation under agitation with Dynabeads for 1 h at 4 °C. The beads were washed five times in ice-cold RIP buffer. RNAzol RT was added to the beads, RNA was purified using the Direct-zol RNA MicroPrep kit (Zymo Research) with DNaseI treatment. Complementary was synthesized using the iScript Reverse Transcript Supermix for RT–qPCR. RT–PCR was run according to the guidelines for TaqMan Fast Advance Master Mix.

RIP from nuclear fractions of cells was performed as previously described (Culjkovic-Kraljacic et al., 2016 (link)). Briefly, 1 mg of nuclear lysate was used for RIP with 7 μg anti-eIF4E antibody (MBL RN001P) or control immunoglobulin G (rabbit, Millipore). After incubation, complexes were eluted by boiling in tris(hydroxymethyl)aminomethane-EDTA containing 1% sodium dodecyl sulfate and 12% β-mercaptoethanol. RNA were isolated using TRIzol reagent and isolated using Direct-zol RNA Microprep Kit (Zymo Research, CA, U.S.A, Cat# R2050).
Note that the complete list of hits from our genome-wide nuclear eIF4E RIP screens is not being provided because the screen was done only once and thus lacks statistical power. However, these results were validated by RT-qPCRs outlined in the text.

Total RNA was extracted from frozen tracheas using the Direct-zol RNA MicroPrep Kit (R2062, Zymo Research) and reverse transcribed to first strand cDNA using the SuperScript™ IV VILO™ Master Mix (Invitrogen). qPCR was performed in a final volume of 20 μL per reaction in 96-well PCR plates using a thermocycler (7500t real-time PCR system, Applied Biosystems). Briefly, 5 μL of cDNA (25 ng) was added to 15 μL of a master mix containing 10 μL of Power SYBR green mix (4367659, Applied Biosystems) and 5 μL of nuclease-free water with the nCoV_IP2 primers (Forward 5’-ATG AGC TTA GTC CTG TTG-3’; Reverse 5’-CTC CCT TTG TTG TGT TGT-3’).