title: GSM4776622: Skeletal_muscle_3; Homo sapiens; RNA-Seq
library_construction_protocol: Since RNA was directly purchased from BioChain for the different tissues, no extraction protocol was needed. 2 μg of DNA‑free total RNA was ribodepleted using Ribo-Zero Gold (Illumina) according to the manufacturer protocol. The resulting rRNA-free RNA was then purified with RNA Clean and Concentrator (RCC) kit (Zymo Research) using a modified protocol to retain RNA ≤ 80 nucleotides (400 μL ethanol 100 % per 50 μL sample). Purified RNA was fragmented 2-4 minutes (depending on the RNA Integrity Number) using NebNext Magnesium RNA Fragmentation Module (New England Biolabs) and once again purified with the RCC kit (Zymo Research) followed by dephosphorylation using T4 Polynucleotide Kinase (Epicentre) and final purification using, again, the RCC kit (Zymo Research). cDNAs were synthesized via TGIRT template-switching with 1 μM TGIRT-III reverse transcriptase (Ingex, LLC) for 15 min at 60℃, during which a DNA oligonucleotide containing the complement of an Illumina Read 2 sequencing primer-binding site became seamlessly linked to the 5' cDNA end. After reaction cleanup, a 5' adenylated DNA oligonucleotide containing the complement of an Illumina Read 1 sequencing primer-binding site was then ligated to the 3' cDNA end with Thermostable 5' AppDNA / RNA Ligase (New England Biolabs). Properly ligated cDNAs were amplified by PCR (12 cycles) to synthesize the second strand and add Illumina flowcell capture and index sequences. Libraries were purified with 2 rounds of Ampure XP beads (Beckman-Coulter) and evaluated on a 2100 Bioanalyzer (Agilent). Libraries were then pooled and sequenced on a NextSeq 500 platform (Illumina) (2 x 75 bp) using a NextSeq 500/550 High Output Kit v2.5 (150 cycles) (Illumina). library strategy: TGIRT-Seq