GSM7593022: Exp12.2_ND4_CCI_CTRL_s1; Homo sapiens; RNA-Seq
Scaffolds were collected and stored at -80˚C. Upon isolation, samples were thawed on ice and incubated with 600µL of Lysis Buffer from RNeasy Kit (Qiagen, Catalog No. 74106). During incubation, samples were cut up using small scissors and continued to sit in a lysis buffer for approximately 20 minutes. The lysed samples were placed onto QIAshredder Mini Spin (Qiagen) columns and spun at 10,000 rpm for 2 minutes. An equal amount of 70% ethanol was added to the supernatant and mixed thoroughly. The ethanol-containing mixture was spun in two rounds through the RNeasy Mini Spin column (Qiagen) at 10,000rpm for 1 minute each; the flow through was discarded. In a new collection tube, 400µL of RNA wash from SurePrep RNA/DNA/Protein Purification Kit (Fisher Scientific, Catalog No. BP2802-50) was added to the RNeasy column and spun at the same setting for 1 minute; the flow through was discarded. After the first RNA Wash, 30µL of DNase and RDD buffer (1:7 ratio) containing solution from the RNase-Free DNase Set (Qiagen, Catalog No. 79254) was incubated on the columns at room temperature for 15 minutes before a second and third round of RNA was performed as well as a final spin to ensure the column was dry. The column was placed into a 1.5mL microcentrifuge tube, and 22uL of RNA Elution Solution (Fisher Scientific) was added to the center of the column and spun at 12,000rpm for 1 minute. Samples were then read on the NanoDrop 2000 spectrophotometer and stored at -80˚C before sequencing. Approximately 1μg of total RNA for each sample underwent RNA quality control analyses before library preparation and bulk mRNA sequencing, as previously described (50, 51). RNA integrity numbers (RIN) were determined with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), which indicated minimal RNA degradation. Illumina Novaseq sequencers generated paired-end reads (150bp) at a read depth of 20 million reads (50). RNA libraries for RNA-seq were prepared by Novogene