Truseq stranded total rna ribo zero gold library preparation kit

Sequencing was carried out at the Institute for Molecular Bioscience Sequencing Facility, The University of Queensland. RNA libraries were made using the Illumina TruSeq Stranded Total RNA (Ribo-Zero GOLD) library preparation kit. Paired-end sequencing was performed using the NextSeq 150 cycle High Output run (2 × 75 bp). A minimum of 25 × 10⁶ reads were obtained for each sample. Sequence reads were trimmed for adapter sequences using Cutadapt and aligned to the mm10 assembly using STAR aligner. The read counts per gene were estimated using RSEM and were utilised to determine differential gene expression between groups using Bioconductor package 'edgeR'. The default TMM normalization method of edgeR was used to normalise read counts between samples. Differentially expressed genes were considered significant if the Benjamini–Hochberg corrected p-value was less than 0.01 and a log2 fold change of > 2 [FC > 2].

We used virally-mediated Cre-mediated recombination in male TrkB^fl/fl mice to induce local TrkB knockdown [4 (link)]. Four weeks after viral injections to the LS, we micro-dissected LS tissue from mice with local LS TrkB knockdown (n = 4) and their GFP controls (n = 4). Brains were extracted and flash-frozen in 2-methylbutane and then sliced on a mouse brain matrix (Zivic Instruments) to generate two coronal brain slabs (1 mm each) at the level of the LS. From each slab, the LS was dissected using a scalpel on a flat metal surface placed over wet ice. Samples were transferred to tubes and placed on dry ice and stored at −80 °C until further processing. Total RNA was extracted from samples using Trizol followed by purification with an RNeasy Micro kit (Qiagen). Paired-end strand-specific sequencing libraries were prepared from 40 ng total RNA using the TruSeq Stranded Total RNA Ribo-Zero Gold Library Preparation kit (Illumina). Following quality control on a BioAnalyzer (Agilent), libraries were sequenced on an Illumina HiSeq 3000. The Illumina Real Time Analysis (RTA) module was used for image analysis and base calling, and the BCL converter was used to generate sequence reads, producing a mean of 135 and a range of 35 to 482 million 100-bp paired-end reads per sample.