Truseq stranded mrna sample prep kit v2

After blood extraction, PBMCs were isolated, and the RNeasy Minikit (Qiagen, Hilden, Germany) was used for total RNA purification. The RNA quantity and quality were assessed by Nanodrop 2000 and 2100 bioanalyzer RNA NANO assay (Agilent Technologies, CA, USA). The samples with an RNA integrity number over 7.5 were selected for sequencing. RNA samples were treated with RNase-free DNase set (Qiagen) following the manufacturer’s instructions. Library generation and sequencing of poly-A RNA were performed at the Centre for Genomic Regulation in Barcelona (Spain). Briefly, 500 nanograms of total RNA were used with Illumina’s TruSeq Stranded mRNA Sample Prep Kit v2 for library synthesis. This process allows capturing polyadenylated coding and non-coding RNAs. Ten libraries were multiplexed and pooled on each line of an Illumina HiSeq2500 sequencer, and a single read of 50 nts (1x50) was performed. We obtained an average of 25 million reads per sample.
Sequences were analyzed with a bioinformatic pipeline described in Supplementary Table 1. Briefly, for the read quality control, FastQC (v. 0.11.8) was used. Then, Trimmomatic (v. 0.38) was implemented for trimming the adapters. Reads were aligned to GRCH38 with STAR (v. 2.6.1d) software, and the Subread package of the FeatureCounts (v. 1.6.4) software was used for gene count estimation.

RNA was isolated with the RNeasy extraction kit (Qiagen, Germantown, MD, USA) and the samples were treated with DNase I (see Supplementary Methods for full details). For T98G cell spike-in normalization, equal numbers of T98G cells for each condition were mixed with a fixed number of D. melanogaster Kc167 cells (1:4 ratio). Libraries were prepared with the TruSeq Stranded mRNA Sample Prep Kit v2 (Illumina, Cambridge, UK) and sequenced with Illumina Hiseq-2500 to obtain 125 bp pair-ended reads. Differential gene expression was assessed with the DESeq2 (1.30.1) package in R, filtering genes that had >10 average normalized counts per million [35 (link)]. For the spike-in libraries, the size factor of each replicate was calculated according to exogenous Drosophila spike-in reads. Expression was considered to be altered when the p-value ≤ 0.05, and the log₂FC was above 0.7 and below −0.7 for up- and downregulated genes, respectively.

Full-thickness skin was collected en bloc as 1 cm × 0.5 cm pieces from three patients with keloids and three individuals with normal skin (control group). In order to compare the superficial dermis levels, de-epithelialization was performed. At the time of surgery, 3.5x magnification loupes were worn by the professionals. The test subjects had no specific underlying diseases such as diabetes or hypertension. RNA was extracted from the keloid and normal tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality of the extracted RNA was evaluated using an Agilent 2100 Bioanalyzer RNA Nano Chip (Agilent Technologies Inc., Santa Clara, CA, USA). The extracted RNA was used to construct RNA libraries using the TruSeq Stranded mRNA Sample Prep kit v2 (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. The quality was analyzed using an Agilent 2100 Bioanalyzer and Agilent DNA 1000 kit. Samples were sequenced using an Illumina HiSeq2500, which yielded an average of 38 million paired-end 100 nucleotide reads.

Total RNA was extracted from three different biological replicates of Drosophila CTRL and GBA-KO brains each, at 3 and 15 days from hatching with TRI Reagent® (Sigma-Aldrich). cDNA libraries were synthesized starting from 500 ng total RNA with TruSeq stranded mRNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) following the manufacturer’s instructions.
Whole transcriptome sequencing was performed on Nextseq500 sequencer (Illumina) at 80 bp read length in paired-end mode, yielding a total of 76 GB and an average of 79,5 million reads per sample. Read pairs were mapped on the Drosophila melanogaster genome reference dm6 using the alignment program hisat2 (http://daehwankimlab.github.io/hisat2/). Raw counts were computed with htseq-count (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287950/) using the Drosophila dm6 annotation Drosophila_melanogaster.BDGP6.95 from ENSEMBL (http://ftp.ensembl.org/pub/release-95/gtf/drosophila_melanogaster/). Raw counts were then normalized as count per million (CPM) by the R-Bioconductor package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html). The same package was adopted to estimate the differentially expressed genes (DEG) between conditions and time points. Upregulated and downregulated biological pathways were evaluated with the web tool FlyEnrichr (https://maayanlab.cloud/FlyEnrichr/).

Total RNA was isolated in triplicates from CHP-212 and HCC-827 cells using the RNeasy Mini isolation kit including on-column DNase digestion according to the manufacturer’s instructions (Qiagen). RNA quality was assessed with the RNA 6000 Nano Kit (Agilent). RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit v2 (Illumina) and sequenced in strand specific paired-end mode, 2x76bp, using the HiSeq2500 platform. Read quality was assessed by running FastQC (version 0.10) on the FASTQ files. Sequencing reads showed high quality, with a mean Phred score higher than 30 for all base positions. A total of 453 billion 76-base-pair (bp) paired-end reads was mapped to the human reference genome (hg38) and the human gene transcripts from Ensembl v76 [31 (link)] by using an in-house gene quantification pipeline [32 ]. On average, more than 97 % of the total reads were mapped to the genome or the transcripts, and more than 91 % to the exons and junctions (expressed reads). Genome and transcript alignments were used to calculate gene counts based on Ensembl gene IDs. The raw RNA-sequencing reads are available in the NCBI Short Read Archive under the accession number SRP062973.