GSM9099648: DGUC Bottom-up_Plasma_2; Homo sapiens; RNA-Seq
Total and small RNA were isolated from samples using the miRNeasy Micro kit (urine and cell culture medium) and miRNeasy Serum/Plasma Advanced kit (blood plasma) according to manufacturer's instructions (cat. no. 217084 and 217204, Qiagen, Valencia, CA, USA), and eluted in 12 µL DNase/RNase-free water. To allow for a proper method comparison during RNA sequencing, synthetic spike-in controls were added as previously described 43,44. In short, per initial amount of biofluid input volume, 2 µL Sequin spike-in control [JV1] (Garvan Institute of Medical Research) and 2 µL RNA extraction Control (RC) spike-ins (Integrated DNA Technologies) were added to the lysate. Following RNA isolation, 2 µL of External RNA Control Control Consortium (ERCC) and 2 µL Library Prep (LP) spike-ins were added. RNA samples were stored at -80 °C until further use. treated to remove residual genomic DNA by adding 1 µL of HL-dsDNase (#70800-202, ArcticZymes, 2 U/µL) and 1 µL reaction buffer (#66001, ArcticZymes) to 12 µL RNA, followed by 10 min incubation at 37 °C, and 10 min incubation at 55 °C to inactivate the DNase. Total RNA sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian (#634487, Takara), including a ribosomal RNA depletion step at the cDNA level, as originally published in 53, with some modifications as reported 54. 8 µL of DNase treated RNA was fragmented for 4 min at 94 °C, followed by the 'highly degraded RNA' workflow. Library quality control was performed with the Fragment Analyzer high sense small fragment kit (Agilent Technologies, sizing range 50-1000 bp). Libraries were quantified using the KAPA Library Quantification Kit (#KK4854, Roche), followed by equimolar pooling and Qubit quantification. The loading concentration of the pool was 0.58 nM with 2% phiX on an S2 100 cycles flow cell (paired-end 2 x 50 nucleotides) on a NovaSeq 6000 instrument (Illumina)