Truseq stranded total rna lt kit

RNA from free and polysomal fractions were extracted using DirectZol kit (Zymo). The sample will be prepared for sequencing in Illumina Platform using TruSeq Stranded Total RNA LT Kit. For clustering and sequencing used TruSeq SR Cluster Kit v3 - cBot – HS and TruSeq SBS Kit v3 - HS (100-cycles). This process generates per reaction, on average, 30 million sequenced fragments of size between 85–100 nucleotides (called reads). The raw data were deposited in the ArrayExpress under the number: E-MTAB-6254.

As starting material, 1 µg RNA from bacteria samples and 1 µg RNA from dual samples (plant + bacteria) were used. Total RNA from bacteria-only samples were depleted of ribosomal RNA (rRNA) by using Ribo-Zero Magnetic Kit (Bacteria) (Epicentre/Illumina). For rRNA removal of plant + bacteria samples, 1:1 mixture of Ribo-Zero Magnetic Kit (Bacteria) and Ribo-Zero Magnetic Kit (Plant Leaf) was used. After rRNA depletion, RNA-Seq libraries were prepared by using the TruSeq Stranded Total RNA LT kit (Illumina). Final complementary DNA (cDNA) libraries were sequenced by using HiSeq2500 at the Core Lab Bioscience Platform (King Abdullah University of Science and Technology [KAUST], Saudi Arabia). Three biological replicates of each sample were used.

Mouse C3H myoblasts were maintained in six-well plates. Myotubes were transfected with siCon or siH19 in triplicates for 40 h following initiation of differentiation. Cells were harvested for RNA extraction 48 h post transfection using the Purelink RNA mini kit (Cat. no. 12183018A). RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA LT kit with Ribo-Zero Human/Mouse/Rat, setA (Cat. no. rs-122–2201) according to the sample preparation protocol. Briefly, 1 μg total RNA was subjected to Ribo-Zero depletion to remove rRNAs. The remaining RNA was purified, fragmented and primed with random hexamers for cDNA synthesis. After first and second cDNA synthesis, cDNA fragments were adenylated and then ligated to indexing adapters. The cDNA fragments were enriched by PCR, purified and then sequenced on an Illumina NextSeq500 using paired-end chemistry and 76-bp cycles. Sequences are available from the GEO with accession number GSE73014.
Illumina BaseSpace (https://basespace.illumina.com/)-embedding tools were used to analyse the RNA-seq data. TopHat Alignment 1.0.0 app was used to map sequencing reads to mm10 genome. Cufflinks Assembly & DE 1.0.0 app containing Cufflinks 2.1.1 and Cuffdiff 2.1.1 was applied to assemble mapped transcripts and calculate differential expression of genes and transcripts.