Truseq rna library preparation kit v2

S. cerealella adult male moths from the control and DAT-fumigated groups were collected and the testis was dissected in a phosphate-buffered saline (PBS) solution under the stereomicroscope. A rubber pad with holes was used to securely hold the centrifuge tube, which was then placed in a container of liquid nitrogen. This setup allowed the centrifuge tube to float in the liquid nitrogen. After dissecting the testis, it was immediately transferred to the pre-cooled centrifuge tube, which was then placed into the liquid nitrogen for preservation. Total RNA was extracted from the homogenates of the testes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and quality of RNA were checked using an Agilent 2100 Bioanalyzer. After DNase I treatment, mRNA was purified from total RNA using magnetic beads with oligo (dT) and fragmented. cDNA was synthesized from mRNA fragments following the protocol for the TruSeq RNA Library Preparation Kit v2 (Illumina, San Diego, CA, USA). After end reparation and adapter ligation, suitable fragments were selected for PCR amplification. Quantification and qualification of the sample library were assessed using an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-time PCR System. cDNA libraries were then sequenced on an Illumina HiSeq™ 2000 platform.

Total RNA from three db/db mice and three db/m mice were isolated using TRIzol^® Reagent (Life Technologies) in accordance with the manufacturer’s instructions, as described previously^{42 (link)}. RNA quality and quantity were measured using an Agilent 2100 Bioanalyzer (Agilent Technologies), and then these RNA samples were used for cDNA library preparation and sequencing, which was conducted by Beijing Genomics Institute (BGI, Shenzhen, China). cDNA libraries were constructed using the TruSeq™ RNA Library Preparation Kit v2 protocol (Illumina), as described previously.⁴⁴ cDNA libraries were sequenced using an Illumina HiSeq2000 sequencer (typical read lengths 90 bp) and 200 bp paired-end reads were generated.

Four samples of the siRNA-transfected HT29 cells were prepared for high-throughput mRNA sequencing. Total RNA of 1 μg was extracted from each sample and the mRNA library (insert size of ~300 bp) was created using the TruSeq RNA Library Preparation kit v2 (Illumina). Paired-end transcriptome sequencing (101 bp read length) was performed on Illumina HiSeq 2500. The number of reads for each sample ranged from 69.4 million to 74.8 million. The raw sequencing data were deposited in the GEO database (Accession Number: GSE81429). RNA-Seq data were aligned to the human genome (hg19 from UCSC) using MapSplice v2.1.7 after standard quality check and trimming with FastQC and Fastx-toolkit, respectively. The mapping rate of reads was between 96.5 and 96.8%. RSEM v1.2.12 was used to estimate the transcriptome abundance for refGene mRNAs. Differentially expressed genes were identified using DESeq2 with the FDR cutoff of 0.05.

To evaluate transcriptome-wide effects of USP22 loss, mRNA-seq and subsequent analysis was performed. SW837, SW480, HCT116, MCF10A and HCC1954 cells were transfected with control or USP22 siRNAs (n = 3–4 per condition). RNA integrity was verified using agarose gel electrophoresis and libraries were prepared using TruSeq RNA Library Preparation Kit v2 (Illumina®) or NEXTflex Rapid Directional RNA-Seq Kit (Biooscientific). Sequencing was performed at the Transcriptome and Genome Analysis Laboratory (TAL, Göttingen) using HiSeq 2000 or 4000 (Illumina®). All mRNA-seq data have been deposited at ArrayExpress (http://www.ebi.ac.uk/arrayexpress, accession numbers: E-MTAB-7393 (HCT116)⁶ , E-MTAB-8214 (SW480), E-MTAB-8215 (SW837), E-MTAB-8247 (MCF10A), E-MTAB-8256 (HCC1954)). Data analysis and Gene Set Enrichment Analysis (GSEA;^{18 (link)}), were performed as previously described^{17 (link),19 (link)}.

Cryopreserved PBMC samples were thawed, viability was assessed using trypan blue (>90% for all samples) and immediately processed for RNA extraction (approximately 7x10⁶ PBMC per sample, range: 4 x 10⁶–11 x 10⁶) with the Qiagen RNEasy Mini kit. RNA quality was assessed by Agilent Bioanalyzer 2100; all samples exhibited RNA integrity numbers (RIN) greater than 8. RNA-Seq libraries were prepared with the Illumina TruSeq RNA Library Preparation Kit v2. Libraries were sequenced in multiplex on the Illumina HiSeq 2500 platform in 100 nucleotide, single-end read configuration (range 3 x 10⁷–7 x 10⁷ total reads per library). Samples from the same patient series were always sequenced in the same multiplex pool to minimize batch effects.
Reads were mapped to the human genome reference (hg19) using the Tophat (v2.0.8b) alignment tool [40 (link)]. Read counts per gene were quantified against Ensembl (v66) transcript reference annotations (appended with gene annotation for IFNL4) using HTSeq-count (v0.5.4p3) [41 (link)].

Thymic tissue samples (n = 4 samples/treatment/line, except n = 3 for the Fayoumi Thermoneutral + LPS group, 31 samples total) were harvested from the same birds used by Monson et al., 2018 and Van Goor et al., 2017 to investigate splenic and bursal transcriptome responses to heat stress and/or LPS^{29 (link),30 (link)}. RNA was isolated from homogenized tissues using the RNAqueous Total RNA Isolation kit (Ambion, Inc.), followed by DNase treatment with the DNA-free™ DNA Removal kit (Ambion, Inc.). Resulting RNA samples were high quality (RNA Integrity Number (RIN) ≥8.3; average RIN = 9.4), as measured on the Eukaryote Total RNA Nano chip (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). Barcoded cDNA libraries were constructed from 0.5 µg total RNA input (TruSeq RNA Library Preparation kit v2, Illumina, Inc., San Diego, CA, USA) and confirmed as high quality using the DNA 1000 chip (2100 Bioanalyzer, Agilent Technologies). Libraries were multiplexed in a randomized block design (n = 8 samples/lane, 4 lanes total, see Supplementary Dataset S1 for lane assignments) and sequenced (100 bp single-end reads) on the HiSeq 2500 (Illumina, Inc.) at the ISU DNA Facility (Ames, IA, USA).