GSM6338775: Patient6; Homo sapiens; miRNA-Seq
Blood samples were collected from LGMD, DMD and FSHD patients and healthy participants in EDTA tubes. Each sample was centrifuged to separate plasma; then, a second centrifugation was performed at 16000g and 4 ºC for 10 minutes. Finally, plasma samples were stored at −80 ºC until RNA extraction. Cell-free total RNA (including miRs) was isolated from 400 µL of plasma using the miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany), following the manufacturer's protocol. RNA was eluted with 25 µL of RNase-free water. The concentration of total cell-free RNA (including miRs) was quantified using NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific Inc, Waltham, MA, USA). Small RNA libraries were generated and indexed using the Lexogen's Small RNA-Seq Library Prep Kit for Illumina sequencing (Lexogen GmbH Campus Vienna Biocenter 5, 1030 Vienna, Austria). Briefly, for library generation 3'-adapter was ligated to RNA and their excess removed by column purification. Next, 5'-adapter was ligated and the RNA, flanked by 5'and 3' adapters, was converted into cDNA. Library amplification was performed to add the complete adapter sequences necessary for the generation of clusters and to assure enough material for quality control and sequencing. External i7 indexes were added during this step to enable multiplexing of libraries for sequencing. The library product was then purified to remove interfering PCR components. Single-end sequencing was performed on Illumina NextSeq550 platform (Illumina, San Diego, CA, USA) on High Output 1x50pb RUN (NextSeq 500/550 High Output v2 75 cycles kit, FC-404-2005).