Rnaeasy minelute kit

Ten microgram of RNA was treated with 2 U of RNase free DNase I (NEB, USA) followed by phenol-chloroform extraction and precipitation. Two μg of DNase treated RNA was polyadenylated using Poly A-tailing kit (Ambion, USA) as per manufacturer’s instructions and column purified with RNAeasy minelute kit (Qiagen, GmBH). Subsequently, each sample was reverse transcribed using 1 μg of linker primer and Superscript III (Invitrogen, USA). Quantitative real time PCR of miRNA sequences was performed on Realplex² mastercycler (Eppendorf, Germany). miRNA validation and profiling was done with miRNA specific forward primer (800 nM), universal reverse primer (800 nM), and a TaqMan probe (200 nM) that binds in the linker primer region, using FastStart Universal Probe Master chemistry (Roche, USA). 5S rRNA sequence was used as endogenous control. In case of target genes DNase free RNA was reverse transcribed using iScript reverse transcription kit (Biorad, USA). Target gene expression profiling was done using gene-specific primers and KAPA SYBR FAST chemistry (Kapa biosystems, USA). Actin was used as an internal reference control. C_T values obtained through qPCR were analyzed using delta delta C_T method to calculate relative fold change values. The details of the primers used in qPCR and other experiments are given in supplementary table S1.

Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s recommendations. Total RNA (100 μg) was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN). mRNA (100–200 ng) was fragmented by hydrolysis (5× fragmentation buffer: 200 mM Tris acetate, pH8.2, 500 mM potassium acetate and 150 mM magnesium acetate) at 94 °C for 90 s and purified (RNAeasy Minelute Kit; QIAGEN). cDNA was synthesized using 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). Double-stranded cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer’s recommendations and purified (Minelute Reaction Cleanup Kit; QIAGEN). Strand-specific rRNA depleted double-stranded cDNA profiling used for the NPC lines was performed with the ScriptSeq kit (catalog number SS10924) from Illumina, according to the instructions of the manufacturer. rRNA depletion was performed with the Ribo-Zero rRNA Removal Kit using 5 μg of total RNA (Human/Mouse/Rat; catalog number RZH110424).

The RNA isolation was performed using a previously described method [45 (link)]. Briefly, total RNA was isolated using Trizol (Invitrogen, MA, USA), and 5 μg of total RNA was purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Furthermore, 100–200 ng of mRNA was fragmented through hydrolysis and purified (RNAeasy Minelute Kit; QIAGEN, Germany). Library DNAs were prepared according to the Illumina TrueSeq protocol using the Truseq Standard mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced by Illumina NextSeq 500 (Illumina, CA, USA) using the Nextseq 500/550 High Output v2.5 Kit (Illumina, CA, USA) to obtain single-end 75 bp reads. The resulting reads were aligned to the mouse genome (mm10) using STAR ver.2.6.0a after trimming to remove the adapter sequence and low-quality ends using Trim Galore! v0.5.0 (cutadapt v1.16). The transcript abundance was determined using RSEM v1.3.1. The counts of gene-expression profiles in those selected datasets were further analyzed through RNAseqChef web-based transcriptome analysis [22 (link)]. Pathway Interaction Database was used for enrichment analysis in RNAseqChef. The Z-scored normalized count is shown in the heatmaps (genefilter: methods for filtering genes from high-throughput experiments. R package version 1.72.1.).