Truseq stranded total rna prep kit

Total RNA was isolated from the ischemic gastrocnemius muscle (n = 4/group) using PureLink^® RNA Kit (Life Technology, Grand Island, NY, USA), as previously described [10 (link),19 (link)]. The quality of RNA was monitored by RNA gel electrophoresis following the manufacturer’s instructions. RNA sequencing (RNA-Seq) was performed as described previously [24 (link)]. Briefly, RNA-seq libraries were constructed from 1 µg of total RNA using the TruSeq Stranded Total RNA prep kit (Illumina, San Diego, CA, USA). The resulting barcoded libraries were sequenced on an Illumina NextSeq 500 using 150-cycle High Output Kits (Illumina). Quality control of the resulting sequence data was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed on 28 November 2021)). All data were analyzed using Illumina BaseSpace applications. Sequencing reads were aligned to the mouse mm10 reference transcriptome and using the TopHat Alignment v1.0.0 application [25 (link)]. Expression values (FPKM; Fragments Per Kilobase of transcript per Million mapped reads) were then generated for each transcript and differentially expressed transcripts (at a false discovery rate (FDR, q-value) of <0.1) were identified using Cufflinks Assembly and Differential Expression v1.1.0 application.

RNA-seq libraries were prepared using TruSeq stranded total RNA prep kit (Illumina Norway AS) targeting the poly-A tail to enrich mRNAs. 15 barcoded libraries (5 control; 5 rIFNg stimulated at 2 h time point; 5 rIFNg stimulated at 24 h time point) were pooled together and sequenced over 2 lanes of HiSeq 3000 (Illumina Norway AS) employing 150 bp paired-end sequencing. Library preparation and sequencing was performed at the Norwegian Sequencing Center, Oslo, Norway.

mRNA libraries for all four experiments were prepared at the Ramaciotti Centre for Genomics (UNSW Australia). The Illumina TruSeq RNA Prep Kit was used for Experiment 1. The Illumina TruSeq Stranded Total RNA Prep Kit was used for Experiments 2-3 and the Illumina TruSeq Stranded mRNA Prep Kit was used for Experiment 4. The six RNA-Seq libraries in Experiment 1 were sequenced on the Illumina HiSeq2000 platform, Experiments 2, 3 and 4 were sequenced using the Illumina NextSeq 500. R1.fastq and R2.fastq files were produced for each sample.

RNA was extracted from tumor samples or Rb cell lines (RB006, RB016, RB018, and wRB6) with or without shESRRG knockdown using a combination of TRIzol and an RNeasy Mini RNA isolation kit (QIAGEN, Valencia, CA, USA). RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Prep Kit with Ribo-Zero Gold to remove cytoplasmic and mitochondrial ribosomal RNA according to the manufacturer’s recommendation (Illumina, San Diego, CA). Total RNA-seq libraries were run on an Illumina NextSeq 500 sequencing instrument according to the manufacturer’s protocol. RNA-seq from the four Rb samples that underwent WGS was obtained with permission from dbGaP (phs000352.v1.p1). Reads were aligned and gene counts were made using STAR, data quality was assessed using FastQC and RSeQC, and gene expression was normalized, batch-corrected, and determined using EdgeR. Significant differences (FDR < 0.05) between cells expressing shESRRG versus shGFP control were calculated using EdgeR. For tumor samples that underwent RNA-seq without a detectable biallelic loss of RB1 seen in exome analysis, RNA tracks were manually curated to look for any RB1 mutations.

Bacteria were grown in M9 mineral medium with 0.4% glucose or LB medium, and samples were prepared at five stages of growth from both cultures to evaluate the span of gene expression. RNA was isolated using the Direct-zol RNA miniprep kit (Zymo, USA) following the manufacturer’s protocol with bead beating and a DNase I treatment protocol. RNA was prepared for sequencing with the NEBNext rRNA depletion kit (E7850; New England Biolabs [NEB], USA), followed by TruSeq Stranded Total RNA Prep (kit from Illumina, USA) following the manufacturer’s protocol. Counts were generated with HTSeq aligned to the respective PGAP annotated genome. Comparisons were done with the Mbale and reference strains aligned to the Mbale annotation. Normalization and exploratory analyses were done with DESeq2 (85 (link)) using the R statistical programming hclust package, which utilizes the complete linkage method with Euclidean distances and is represented using the pheatmap package in R.