Nextseq 2000 machine

Total RNA extraction was performed by using TRIzol reagent (Invitrogen, #15596026) from 7-day-old seedlings, and dissolved in DEPC-treated H2O. Total RNAs were used for library preparation with Real Seq Bioscience RealSeq^R-AC Kit (500–00012). The final library products were further purified using 6% polyacrylamide gel (Novex™ TBE Gels, EC6265BOX). The 145–160nt products were excised from the gel for sequencing (single end 50 bp) on a NextSeq 2000 machine (Illumina). The small RNA sequencing data were trimmed using Trimmomatic (v.0.39) and then mapped to the pseudo-genome sequence with insertion of 2 × 180 or 5 × 180 in TAIR10 and called small RNA using ShortStack version 3.8.5 (44 (link)) with parameter setting "–mincov 1rpm –pad 75 –mismatches 0 –nohp.

RNA was isolated using TRIzol and subsequently DNase treated. Integrity of isolated RNA was evaluated using RNA ScreenTapeon the Agilent Tapestation (Agilent Technologies). Sequencing was performed on an Illumina NextSeq 2000 machine at the Sequencing Facility, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research using standard protocols. Briefly, 16 mRNA sequencing samples were pooled and sequenced on NextSeq 2000 P2 using Illumina Stranded mRNA Ligation Kit and paired-end sequencing. The samples had 56 to 69 million pass filter reads with more than 95% of bases above the quality score of Q30. The RNA sequencing (RNA-seq) data for the human breast cancer cell lines were deposited in the NCBI's GEO database under accession number GSE223181.

RNA extraction of in vitro cultured Th17 cells was performed with the NucleoSpin RNA XS Kit (Macherey-Nagel) according to the manufacturer’s protocol. RNA concentrations and integrity were measured using a RNA 6000 NanoKit (Agilent) on a 2100 Bioanalyzer (Agilent). Sequencing was performed by the Sequencing Platform of the Luxembourg Center for Systems Biomedicine (LCSB) of the University of Luxembourg. Samples were prepared using an Illumina Stranded mRNA library prep kit (Illumina) with the addition of IDT for Illumina DNA/RNA UD indexes (Illumina). Paired-end sequencing was executed using an Illumina NextSeq2000 machine with a read length of 2 × 50 bp.
RNA-seq transcript alignment was performed with Salmon (Patro et al., 2017 (link)) against the Mouse Transcriptome from Genecode release M30 assembly GRCm39 (Frankish et al., 2018 ). Subsequent analysis was conducted in R, and Tximeta (Love et al., 2020 (link)) was used to assign transcripts to genes before differential analysis with DESeq2 (Love et al., 2014 (link)). Gene set enrichment analysis (GSEA) was performed using ClusterProfiler (Yu et al., 2012 (link)).