Truseq stranded total rna sample preparation guide

QIAamp RNA Blood Mini Kit was used for purification of total RNA from the whole blood. During the QIAamp procedure for purification of RNA from blood, erythrocytes are selectively lysed, and leukocytes are recovered by centrifugation. The leukocytes are then lysed using highly denaturing conditions that immediately inactivate RNases, allowing the isolation of intact RNA. After homogenization of the lysate by a brief centrifugation through a QIA shredder spin column, ethanol is added to adjust the binding conditions, and the sample is applied to the QIAamp spin column. RNA is bound to the silica membrane during a brief centrifugation step. Contaminants are washed out, and total RNA is eluted in 30 μL or more of RNase-free water.
RNA concentrations were measured using Qubit 3.0 (Invitrogen, Carlsbad, CA, USA). The RNA quality was verified on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). High-quality (RIN > 8) RNA samples were processed for library preparation. Further sample preparation was conducted by Illumina TruSeq^® Stranded Total RNA sample preparation guide.

TRIzol reagent (Invitrogen, CA, USA) was used for RNA extraction, and cDNA libraries were prepared based on the TruSeq^® Stranded Total RNA Sample Preparation Guide (Illumina, Part # 15031048, San Diego, CA, USA). Equal concentrations of each library were sequenced using a NextSeq 500 (Illumina) platform to create pair-end 75-bp reads. Quality assessment and trimming of the generated sequences were done by the RNA-seq alignment tool from BaseSpace (Illumina), followed by alignment to the human reference genome (hg38) with STAR 2.5.2b [21 (link)]. The expression levels of genes in each sample and the corresponding fold changes were estimated by DESeq2 1.14.1 (Partek Genomics Suite, St. Louis, MO, USA) [22 (link)] with GENCODE V19 (V25) annotation [23 (link)]. Relative expression of each gene is represented by CPM (Counts Per kilobase Million). Partek Genomics Suite and statistical package were used for the statistical analysis, hierarchical clustering differential expression analysis, and Gene ontology (GO) enrichment. Canonical pathway analysis was performed by using the Ingenuity Pathway Analysis (IPA).

For all eight whole RNA samples, rRNA depletion, cDNA library preparation and metatranscriptome sequencing were performed at the DOE JGI. Library preparation was performed following the TruSeq Stranded Total RNA sample preparation guide by Illumina (San Diego, CA, USA). Prior to library generation, rRNA depletion was performed using the Ribo‐Zero rRNA Removal Kit (Bacteria) (Epicentre, Chicago, IL, USA). For library construction, the TruSeq Stranded mRNA Sample Preparation Kit (Illumina; San Diego, CA, USA) was used. Depleted mRNA was fragmented and reverse‐transcribed using the Superscript II reverse transcriptase (Invitrogen, Waltham, MA, USA). After second‐strand synthesis, end‐repair, A‐tailing, adapter ligation of the double‐stranded cDNA and 10 cycles of PCR amplification followed.
Quantification of metatranscriptome libraries was performed using the next‐generation sequencing library qPCR kit (Kapa Biosystems, Wilmington, DE, USA) and the LightCycler 480 real‐time PCR instrument (Roche, Basel, Switzerland). Sequencing preparation was performed using the TruSeq paired‐end cluster kit (v3, Illumina; San Diego, CA, USA). Finally, metatranscriptome sequencing was performed on the HiSeq 2000 sequencer using the TruSeq SBS sequencing kits (v3) following the 2 × 150 indexed high‐output run instruction (both by Illumina).