Truseq mrna v2 kit

Isolated RNA at a concentration of 20 ng/µl was further purified with a TruSeq mRNA v2Kit (polyA, Illumina). Biological triplicates were sequenced on a HiSeq2500 using paired-end chemistry and 2×125 cycles. The sequence depth was ∼100 million reads per sample. Runs were performed by deCODE Genetics.
To assess the differentiation state of generated CNs, cross-platform comparisons with spatiotemporal data from the developing human brain of the BrainSpan atlas were performed by pairwise comparison of generated CNs with each BrainSpan atlas sample. By ranking gene expression for each pairwise comparison, rank difference values for all genes were used to calculate Spearman’s rank correlation coefficients. A Wilcoxon rank-sum test was applied if a category of interest (neocortex, subcortex, ganglionic eminence or cerebellum) had significantly higher Spearman’s correlation coefficients than the background of all paired correlations. –Log 10 P-values of significant differences of CNs and spatiotemporal brain data were shown in heat maps.

For studies with BIN67, SCCOHT-1, and COV434 cells, RNA from frozen cell pellets was extracted using the Zymo Quick-RNA Miniprep Kit (Zymo cat# R1054). Total RNA was prepared using the Illumina TruSeq mRNA v2 kit with an input of 500 ng of RNA for each sample to produce unstranded RNA libraries following the manufacturer’s protocol. Final RNA libraries were quantified using the Qubit High Sense Reagent kit and Agilent Tapestation HSD1000 tapes. Libraries were equimolar pooled and paired end sequenced across three lanes of a HiSeq4000 (paired end x 78 bp).
For RNA-seq analysis of the BIN67 pIND20-A-FOS and A427 pIND20-BRG1 cell lines, RNA was extracted from frozen cell pellets in biological duplicate (BIN67 pIND20-A-FOS) and biological triplicate (A427 pIND20-BRG1) using Zymo Quick-RNA Miniprep Kit. Library preparations and RNA sequencing was performed by Novogene Inc.

Cell pellets for Sdhc fl/fl and Sdhc fl/+ iMEF lines collected over the time series experiment were subjected to RNA extraction using the Qiagen RNeasy kit. Purified RNA was the submitted to the Mayo Clinic Medical Genome Facility for indexing and preparation of deep sequencing libraries using the Illumina TruSeq mRNA v2 kit, followed by deep sequencing on a HiSeq 4000 instrument, multiplexing 8 samples per lane and performing 100 sequencing cycles. Following sequencing, reads belonging to individual experiments were deconvoluted on the basis of unique sequence barcodes. Deconvoluted sequence read data for individual experiments are available from the NCBI sequence read archive (SRA) under identifier SRP117182.
Paired end sequence reads were then aligned to the mm9 mouse reference genome using the Bowtie fast read aligner [28 ] and the Mayo Clinic Research Computing Facility Beowulf-style Linux cluster. SAM files were then converted to BAM file format using SAMtools [37 (link)], and FPKM quantitation of individual transcript abundance was performed using R package systemPipeR [38 (link)], available through the Bioconductor project. The resulting processed gene expression datasets are available via NCBI Gene Expression Omnibus (GEO) under entry GSE103662.

We surface-sterilized embryos from one freshwater and one oceanic cross and assigned 20 individuals from each clutch to Pseudomonas monoassociation (MA) or conventional (CV) microbiota treatments, as described above. At 14 dpf, we euthanized the larvae using MS222, dissected the GI tract from the stomach to the urogenital opening, and isolated total RNA using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the protocol described by Leung and Dowling (2005 (link)). We used 200 ng of total RNA from each of four stickleback guts per family-treatment combination (16 total) to generate RNA-seq libraries with the TruSeq mRNA v2 Kit (Illumina, San Diego, CA). Ten million 100-nucleotide (100-nt) Illumina HiSeq2500 reads from each sample were aligned to the Ensembl v75 threespine stickleback reference genome; we counted the number of reads mapped uniquely to each gene model, and we performed normalization and differential expression analysis using the negative binomial generalized linear models implemented in the R statistical package edgeR (Robinson et al., 2010 (link)). The RNA-seq data are part of a larger gene-expression study and will be described fully in a future publication.

The transcriptome’s raw reads from the venom gland of Pamphobeteus verdolaga were obtained from the European Nucleotide Archive (ENA) under accessions numbers: PRJEB21288/ERS1788422/ERX2067777-ERR2008012. As explained by Estrada and co-workers [32 (link)], two female specimens were collected from the province of Antioquia, Colombia, under the contract 155 signed by the University of Antioquia and the Environmental Ministry of Colombia, and the venom glands were extirpated. The total RNA was obtained through TRIzol^® reagent (ThermoFisher Scientific, Waltham, MA, USA) while the purification process of mRNA and the library creation was carried out with the Illumina mRNA TruSeq kit v2, as indicated by the manufacturer. The library 100 bp pair-ended reads was sequenced in an Illumina Hiseq 2500 instrument (Illumina Inc., San Diego, CA, USA).