title: GSM6132172: Disease,vastus lateralis muscle, patient 12; Homo sapiens; RNA-Seq
library_construction_protocol: We used the same protocol for RNA extraction from muscle tissue as the one described by Cummings et al. RNA was extracted from the 93 muscle biopsies using the Direct-zol RNA Miniprep Plus kit ZY-R2073 (Zymo Research, Irvine, CA, USA) according to the kit's instructions. In order to avoid technical variability, all samples were extracted with the same kit on two consecutive days in the same lab by the same  lab technician. Ninety-three RNA samples were treated with DNase prior to their submission to the VIB Nucleomics Core (www.nucleomics.be). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). RNA Integrity Number (RIN) scores mostly varied from 6.3 (partially degraded) to 8.4, except for a few samples that were highly degraded. Per sample, an amount of 5 ng of total RNA (or 7µl RNA when 5 ng was not available) was used as input for the SMART-Seq Stranded Kit (Cat. No. 634444; protocol version “022819”; low input option; Takara Bio USA, San Jose, CA, USA). This kit can deal with degraded as well as high-integrity input RNA. Positive and negative controls were included in the experimental design using 5 ng control RNA (included in the kit) and 7µl RNase-free water, respectively.First, RNA is converted to cDNA using random priming (scN6 Primer) and SMART (Switching Mechanism At 5' end of RNA Template) technology (Takara Bio USA, San Jose, CA, USA) and then full-length adapters for Illumina sequencing (including specific barcodes for dual-indexing libraries) are added through PCR using a limited number of cycles (5 cycles). The PCR products are purified and then ribosomal cDNA is selectively depleted by cleaving the ribosomal cDNAs by scZapR in the presence of mammalian-specific scR-Probes which target nuclear and mitochondrial rRNA sequences. This process leaves the library fragments originating from non-rRNA molecules untouched. The remaining cDNA fragments are further amplified with primers universal to all libraries (14 cycles). Lastly, the PCR products are purified once more to yield the final cDNA library. All libraries were finally quantified using Qubit dsDNA HS kit (Thermo Fisher Scientific, Waltham, MA, USA) and their size distribution was checked using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Nine libraries were excluded from downstream sequencing as there was no yield: control C6 (VL), control C8 (SM), patient 6 (SM), patient 7 (SM), patient 8 (VL), patient 12 (RF), and patient 16 (SM, VL, RF) (Supplementary tables 1 and 2).