Sureselect strand specific rna library prep kit

RNA was extracted from the lungs of Yap^f/f; Shh^Cre/+ embryos and their wild-type littermates at 12.5 and 14.5 dpc using TRIzol (Life Technologies) and the RNeasy Kit (Qiagen [Hilden, Germany]). RNA quality was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies [Santa Clara, CA, USA]).
Paired-end libraries were prepared using the SureSelect Strand-Specific RNA Library Prep kit (Agilent Technologies). Multiplexed sequencing was run in a HiSeq2000 or HiSeq4000 sequencer (Illumina [San Diego, CA, USA]). Read alignment and differentially expressed genes were analyzed by the Maverix Biomics (San Mateo, CA, USA) Analytic Platform (Maverix). Functional enrichment analysis was performed using Ingenuity Pathway Analysis software (version 7.1). Datasets are deposited on the Gene Expression Omnibus database under the accession number GSE93339 (GEO,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93339).

RNA was extracted from fresh frozen tissues using NucleoSpin RNA (Takara, Shiga, Japan). Quality control was performed via Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and all RIN values were over 8.0. RNA-seq was performed as described previously [6 (link)]. RNA library was prepared by using SureSelect Strand Specific RNA Library Prep Kit (Agilent, Palo Alto, CA, USA) and 100 bp paired end RNA-seq was performed via HiSeq2500 (Illumina, San Diego, CA, USA). Ribosomal RNA and adopter sequence were deleted from FASTQ data. RNA-seq reads were aligned to the human genome assembly hg19 using TopHat v2.1.0 with fusion-search option based on Bowtie algorithm and fusion candidates were selected with the following requirements: each read mapping across a fusion point have ≥13 bases matching on both sides of the fusion, with no more than two mismatches; and the two ‘sides’ of the candidate transcripts either reside on different chromosomes or reside on the same chromosome and are separated by ≥100 kbp [64 (link)].

Frozen muscle tissues were ground using the Geno/Grinder^® 2010 (SPEX™, Metuchen, NJ, USA). Ground tissue was homogenized in Trizol^® (Life Technologies™) and RNA extracted using the Trizol^® plus RNA extraction kit (Life Technologies™) according to the manufacturer’s instructions. RNA quality and integrity were evaluated by the 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). All samples with an RNA integrity number larger than 7 and a 28S/18S ratio higher than 1.0 were used for library preparation using the SureSelect Strand Specific RNA Library Prep Kit (Agilent Technologies). The cDNA library was constructed using the Illumina TruSeqTM RNA Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. RNA-seq was performed on the Illumina NovaSeq 6000 platform using a pairing end strategy (read length 150 base pairs (bp)).

Livers from control or Lis1 KO mice were dissected and RNA was extracted using RNAiso Reagent (TaKaRa). RNA quality was evaluated using the Agilent 2200 Bioanalyzer (Agilent Technologies). Paired-end libraries were prepared using the SureSelect Strand-specific RNA Library Prep kit (Agilent Technologies). Multiplexed sequencing was run in a HiSeq2500 sequencer (Illumina). Read alignment and gene expression levels were analyzed by the Maverix Analytic Platform (Maverix). The data set was culled from differentially expressed genes with a cutoff of at least a 2-fold change in expression levels (p value ≤0.05). The pathway enrichment analysis and the network analysis were performed on differentially expressed genes using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA). The data were deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE108096).

We prepared 150–200-bp insert libraries from total RNA using the SureSelect Strand Specific RNA Library Prep Kit (Agilent Technologies) together with the TruSeq stranded mRNA Sample Prep Kit or the TruSeq mRNA-Seq sample preparation kit (Illumina). The libraries were sequenced using 100-bp PE sequencing on HiSeq 2500, HiSeq2000, or GAIIx (Illumina) according to the manufacturer’s instructions.
The gene fusions were identified using our in-house pipeline^{48 (link)} and fusion (https://github.com/Genomon-Project/fusion), enabling a valid selection of putative chimeric transcripts generated by the STAR^{49 (link)} algorithm, as previously reported^{50 (link)}.
RNA-seq reads were aligned to the human transcriptome (UCSC gene) and genome (GRCh37/hg19) references using BWA to calculate gene expression. After the transcript coordinates were converted to genomic positions, an optimal mapping result was selected from either the transcript or genome mapping by comparing the minimal edit distance to the reference. Local realignment was performed using an in-house short-read aligner with a smaller k-mer size (k = 11). Finally, fragments per kilobase of exon per million fragments mapped (FPKM) values were calculated for each UCSC gene while considering strand-specific information.

RNA was extracted from peripheral white blood cells using RiboPure Blood Kit (Ambion) according to manufacturer’s instructions. RNA was extracted from milk somatic cells using Trizol Plus RNA Purification Kit (Ambion) according to manufacturer’s instructions. RNA quality was assessed using the BioAnalyser 2100 (Agilent Technologies, Palo Alto, CA) and concentrations were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE).
RNAseq libraries were prepared using the SureSelect Strand Specific RNA Library Prep Kit (Agilent) according to manufacturer’s instructions. Each library was uniquely barcoded, randomly assigned to one of four pools and sequenced on a HiSeq 3000 (Illumina Inc) in a 150-cycle paired-end run. One hundred fifty base paired-end reads were called with bcltofastq and output in fastq format. Sequence quality was assessed using FastQC. QualityTrim (https://bitbucket.org/arobinson/qualitytrim) was used to trim and filter poor quality bases and sequence reads. Adaptor sequences and bases with quality scores less than 20 were trimmed from the end of reads. Reads with mean quality scores less than 20, or greater than 3 N, or greater than three consecutive bases with a quality scores less than 15, or final length less than 50 bases were discarded. Only paired reads were retained for alignment.