Truseq stranded mrna library prep kit v2

Kidneys and hearts of DO mice were processed as follows. The pulverized samples were lysed and homogenized in Ambion TRIzol reagent (15596026, Thermo Fisher Scientific). Total RNA was isolated using miRNeasy Mini Kit (217004, QIAGEN) according to the manufacturer’s protocol with the optional deoxyribonuclease digest step. Polyadenylate [poly(A)] + RNA-seq libraries were generated using the TruSeq Stranded mRNA Library Prep Kit v2 (RS-122-2001, Illumina). Libraries were pooled and sequenced 100–base pair (bp) single-end on the HiSeq 2500 (Illumina) using TruSeq SBS Kit v4 reagents (Illumina) at the Jackson Laboratory (Bar Harbor, ME).
The LBR-null kidneys were lysed and homogenized in TRI Reagent (R2050, Zymo Research). TRIzol RNA Miniprep Plus Kit (R2070, Zymo Research) was used to isolate total RNA. Library preparation with poly(A) selection and 150-bp paired-end sequencing on an Illumina HiSeq 2500 were performed by GENEWIZ (South Plainfield, NJ).

mRNA libraries were constructed using a TruSeq Stranded mRNA Library Prep kit V2 (Illumina) and were paired-end sequenced on a HiSeq 4000 system (Illumina) at the NCI Frederick Sequencing core facility.
RNA-Seq reads were processed using the Snakemake-based lcdb-wf transcriptomics pipeline v1.3c (https://github.com/lcdb/lcdb-wf) (52 (link)). Briefly, quality was assessed using FastQ Screen and FastQC, and adapter sequences and low-quality bps were trimmed using cutadapt. Trimmed reads were then mapped to the human reference genome (gencode v30) using HISAT2 (v2.1.0), and transcript levels were quantified using the featureCounts method from RSubRead (v1.6.4). Differential expression analysis was performed in the R/Bioconductor environment (R v4.0.2; Bioconductor v2.48.0) using the DESeq2 package (v1.28.0) (53 (link), 54 (link)). After constructing a DESeqDataSet object from the read counts generated by RSubRead, generalized linear models were fit with default parameters using a treatment coding design (e.g., NHWD870 versus vehicle). Finally, shrunken log-fold changes were estimated for each contrast using the apeglm adaptive t prior shrinkage approach (55 (link)).