Moloney murine leukemia virus reverse transcription

Total RNA prepared from WT DT40 cells or various KOs (∼5 × 10⁶ cells) and from WT HCT116 cells or various KOs (∼3 × 10⁶ cells) by the acid guanidinium/phenol/chloroform method using ISOGEN (Nippon Gene) was converted to cDNA using Moloney murine leukemia virus reverse transcription (Invitrogen) and random primers. Full-length open reading frame of gEDEM1, gEDEM2, gEDEM3, gERmanI, hEDEM1, hEDEM2, or hEDEM3 was amplified using PrimeSTAR HS DNA polymerase (Takara Bio Inc.) and a pair of primers, namely, 5′-GGGTACCATGCAATGGCGCTCGCTGGT-3′ and 5′-CTCTAGAGATCAAGCCCACCATCCGAT-3′ for gEDEM1, 5′-GGAATTCACCATGGCGCTGCTGCGCTCGCT-3′ and 5′-GCAGATCTGGAGTGTTGTCCAGGAAGACCT-3′ for gEDEM2, 5′-GGAATTCACCATGGGCGGAGCTGCGGGCTG-3′ and 5′-CTATCGATGGTAGTTCATCCTTTTCCATCA-3′ for gEDEM3, 5′-GAAGATCTCGCCATGTACGCTGCCGCCGCC-3′ and 5′-GCTCTAGATGCTGGCACCCAGATGGGGAG-3′ for gERmanI, 5′-GAAGATCTGACCATGCAATGGCGAGCGCTC-3′ and 5′-CGGGATCCAATCAAACCAACCATCTGGTC-3′ for hEDEM1, 5′-CCCAAGCTTGCTCTATGCCTTTCCGGCTGC-3′ and 5′-GCTCTAGATGAGGAGTCTAGGAAAACCTG-3′ for hEDEM2, or 5′-GAAGATCTGGCCATGAGCGAAGCCGGCG-3′ and 5′-CGGGATCCTAGCTCATCCTTCTCCATCA-3′ for hEDEM3.

Total RNA was extracted from in vitro cultured BMMs using Trisure (Bioline). cDNA synthesis was performed with Moloney murine leukemia virus reverse transcription (Invitrogen) at 38°C for 60 min. Real-time PCR was performed using SYBR Green PCR Master Mix in an Applied Biosystems StepOnePlus detection system. The fold difference in mRNA expression between treatment groups was determined by a standard ΔΔCt method. β-actin was analyzed as an internal control. The primer sequences of individual genes are listed in Table S1.

Total RNA prepared from WT DT40 cells or various KOs (∼5 × 10⁶ cells) and from WT HCT116 cells or various KOs (∼3 × 10⁶ cells) by the acid guanidinium/phenol/chloroform method using ISOGEN (Nippon Gene) was converted to cDNA using Moloney murine leukemia virus reverse transcription (Invitrogen) and random primers. The full-length open reading frame of gEDEM1, gEDEM2, gEDEM3, hEDEM1, hEDEM2, or hEDEM3 was amplified using PrimeSTAR HS DNA polymerase (Takara Bio Inc.) and a pair of primers described previously (Ninagawa et al., 2014 (link)).

Cells were sorted into either Trizol LS reagent or complete medium and lysed with Trizol reagent. RNA was extracted using miRNeasy (Qiagen) and treated with DNase I (Qiagen), according to the manufacturer’s instructions. Quantities of extracted RNA were determined using NanoDrop2000 for cDNA synthesis or Agilent Bioanalyzer 2100 before RNA-Seq Library preparation. All RNA sent for library preparation qualified for RNA integrity number >8.
cDNA synthesis was performed using Moloney murine leukemia virus reverse transcription (Invitrogen) in the presence of RNase inhibitor (Promega). qRT-PCR primers used in this study are listed in Table S1. qRT-PCR was performed using SYBR Green MasterMix (Applied Biosystems) and QuantStudio 7 Flex Real-Time PCR System.

CXCR5⁺ CD4 cells were sorted on a FACSAria (BD) from PBMCs. RNA was extracted using the RNeasy Mini Kit (Qiagen) and converted to cDNA. Random primers and Moloney murine leukemia virus reverse transcription (Invitrogen) were used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7900HT with Applied Biosystem predesigned TaqMan gene expression assays and reagents according to the manufacturer’s instructions. The following probe was used (identified by Applied Biosystem assay identification number): P2RX7 (Hs00175721_m1). For each sample, mRNA abundance was normalized to the amount of TAT-box binding protein (Hs00427620_m1), used as gene reference, and expressed as arbitrary units.

RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific), and cDNA synthesis was performed using Moloney murine leukemia virus reverse transcription (Thermo Fisher Scientific). qRT-PCR (in triplicate) was performed using Power SYBR Green master mix (Thermo Fisher Scientific) using a 7900HT Fast Real-Time PCR machine (Applied Biosystems). The following forward and reverse primers were used: Cdkn1a/p21 (5′-CAC​AGC​TCA​GTG​GAC​TGG​AA-3′, 5′-ACC​CTA​GAC​CCA​CAA​TGC​AG-3′), Bbc3/Puma (5′-GCG​GCG​GAG​ACA​AGA​AGA-3′, 5′-AGT​CCC​ATG​AAG​AGA​TTG​TAC​ATG​AC-3′), RhoD (5′-ATT​GTT​GTG​GGC​TGC​AAG​ATA-3′, 5′-CGA​GCT​GAA​CAC​TCA​AGA​TAG​G-3′) and Actb (5′-TCC​TAG​CAC​CAT​GAA​GAT​CAA​GAT​C-3′, 5′-CTG​CTT​GCT​GAT​CCA​CAT​CTG-3′). Transcript abundance was calculated using a standard curve and normalized to Actb.