Mrna seq sample preparation kit

A ∼10 cm $${}^2$$ large piece of a D. vexillum colony was collected on 14 December 2009 from the upside of a settlement plate that was deployed about six months earlier on 25 March 2009 at a depth of 1 meter from the south pier of the islet Hompelvoet (Grevelingen, The Netherlands) in an enclosed marine lake with minimal tidal differences. One piece of this colony was used for the first draft in 2016 [26 (link)], while another piece of the same colony was used for transcriptome analyses. This piece was preserved in RNAlater (Ambion) at -20  $${}^{\circ }$$ C prior to RNA extraction and subsequent sequencing in February 2010. Total RNA was extracted using the RNeasy kit according to manufacturer’s instructions (QIAGEN GmbH, Hilden). A transcriptome library was prepared from 10 mg total RNA, using the Illumina mRNA-Seq Sample Preparation Kit according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). The mRNA-Seq library with a read length of 2 × 76 nucleotides was sequenced using the next generation sequencing apparatus Illumina GAIIx according to the manufacturer’s description at ZF-Screens.

Total RNA was extracted from B. glumae BGR1, BLONN (lon::Gm), and BLONC (lon::Gm/lon) grown in LB medium at 37°C for 10 h after subculture using RNeasy mini kits (Qiagen) following the manufacturer's protocols. Extracted total RNA was treated with RNase‐free DNase I (Ambion) to remove DNA. The quantity and quality of the total RNA were evaluated using RNA electropherograms (Agilent 2100 Bioanalyzer) and by assessing the RNA integrity number. From each sample with an RNA integrity number value greater than 8.0, 8 μg of total RNA was used as starting material and treated with the MICROBExpress mRNA Enrichment kit (Invitrogen). The resulting mRNA samples were processed for the sequencing libraries using the Illumina mRNASeq Sample Preparation kit (Illumina) following the manufacturer's protocols. One lane per sample was used for sequencing by the Illumina Genome Analyzer IIx (Illumina) to generate nondirectional, single‐ended, 36‐base‐pair reads. Quality‐filtered reads were mapped to reference genome sequences (NCBI BioProject accession: PRJNA59397 ID: 539397, http://www.ncbi.nlm.nih.gov/bioproject/59397) using the BWA package (Li & Durbin, 2009 (link)). The mRNA reads were normalized to reads per kilobase per million mapped reads (Mortazavi et al., 2008 (link)). The NCBI SRA accession number for the RNA sequencing data series of BGR1, BLONN, and BLONC is PRJNA727974.

Total RNA was extracted from the testes of 20-, 75- and 270-day-old Duroc and Meishan boars (designated D20, D75, D270, M20, M75 and M270) using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The samples were sent to Shenzhen Genomics Institute (BGI) with drikold. The RNA integrity and concentration were evaluated with a NanoDrop 6000 Labchip kit (Nanodrop, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). An RNAeasy MiniElute Cleanup kit (Qiagen, Valencia, CA, USA) was used for RNA purification to obtain six RNA pools. Construction of the sequencing libraries was performed using the Illumina mRNA-seq Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Sequencing was performed using the Illumina Genome Analyzer (Illumina, San Diego, CA, USA). Approximately 70–83 M clean reads per sample were generated for the genome-wide transcriptomic analyses.