Qiaseq stranded rna library kits

RNA was extracted from 1 × 10⁷ cells with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and the concentration was measured with a Qubit 3.0 Fluorometer. For NGS, 1 µg of RNA was treated with the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme) according to the manufacturer’s protocol and the concentration was measured again. Subsequently, 10–100 ng of rRNA depleted RNA was used to prepare RNA-seq library with QIAseq Stranded RNA Library Kits (Qiagen). Size distribution of DNA fragments of final libraries were confirmed using an Agilent Fragment analyzer with the DNF 474 kit and libraries were quantified with a Qubit 3.0 Fluorometer and the KAPA library quantification kit. All the DNA libraries were pooled and sequenced on the Illumina NextSeq 500 platform.
For RT-qPCR, extracted RNA was subjected to DNase I (NEB) treatment and purified with Agencourt® RNAClean™ XP (Beckman Coulter) before first-strand synthesis. First-strand synthesis was carried out by using Superscript III Reverse Transcription System (Thermo Scientific) according to the manufacturer’s protocol. cDNA was then analyzed by qPCR on LightCycler® 480 Instrument II. Primers used in this study are listed in Supplementary Data 4.

The RNA-seq data were obtained from the Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA). RNA library preparation was performed as described in the instructions provided in the QIAseq Stranded RNA Library Kits (QIAGEN, Dusseldorf, Germany). Total RNA was extracted from cells, and the mRNA was fragmented to an average insert size of 200–400 bp. The resulting RNA fragments were then converted into first-strand cDNA using reverse transcriptase (Thermo Fisher Scientific, Massachusetts, USA) and random primers. The first-strand cDNA was further converted into double-stranded DNA in the presence of dUTP, and these cDNA fragments were subjected to the addition of a single ‘A’ base and subsequent ligation of the adapter. The products were purified and enriched via PCR to generate the final library. The libraries were sequenced on the Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA). Raw sequences were mapped to the mouse genome mm10 by STAR (v2.5.4b_ENREF_32) [12 (link)]. The expression level FPKM values were obtained from Cuffnorm in the Cufflinks package (v2.2.1) [13 (link)]. The data could be assessed online at the Gene Expression Omnibus (GEO) (No. GSE205156).