title: GSM8840293: GE42.female_4027_PRE_novice; Homo sapiens; RNA-Seq
library_construction_protocol: Exosomes were isolated from human plasma samples (300-500 µl) by centrifugation at 300 x g for 10 min at 4°C to remove cell debris. Supernatants were transferred to microcentrifuge tubes, filled with 1x PBS, balanced for mass, placed in a pre-cooled Beckman Coulter Type 70.1 rotor, and centrifuged at 10,000 × g for 30 min at 4°C with maximum acceleration and gradual deceleration. Supernatants were transferred to new tubes, and exosome pellets were resuspended in 100 µl 1x PBS. Samples underwent another round of ultracentrifugation at 100,000 × g for 2 hrs. at 4°C to wash the exosome pellet. The final exosome pellets were resuspended in 150 µl 1x PBS, analyzed by nanoparticle tracking using the same protocol as with whole plasma, and stored at -80°C. Total RNAs were purified from exosome pellets using Direct Zol RNA mini kit (Zymo Research). RNAs were eluted in 20 µl of RNAse-free water and concentrated to 5 µl using SpeedVac Vacuum Concentrator (Thermo Fisher Scientific). A total of 44 to 523 ng of exosome-specific extracellular small RNAs (exRNAs) were obtained from 28 human plasma samples. exRNA quantity and integrity were determined using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), respectively. Small exRNA libraries were generated using NEXTFlex Small RNA-seq Kit v4 with UDIs (Revvity Inc, Waltham, MA, USA) according to modified manufacturer's protocol to account for low exRNA concentrations in exosome samples. The strategy for small RNA libraries aimed at a range of 16-120 nucleotide (nt)-long RNA of transcripts for accurate identification and quantification of exosome-specific extracellular microRNAs, ncRNAs, and protein-coding RNAs. NEXTFLEX® 3' Adenylated Adapter v4 was ligated at 25°C for 1h. Following 3' Adenylated Adapter inactivation, NEXTFLEX 5' Adapter v4 was ligated at 20°C for 1h. Reverse Transcription-First Strand Synthesis from 5' and 3' NEXTFLEX® Adapter Ligated RNA templates was conducted at 50°C for 1h, followed by inactivation at 90°C for 5 min. First Strand Synthesis products were purified using NEXTFLEX Cleanup Beads by two washes in 80% ethanol. RNA templates were combined with NEXTFLEX UDI Barcoded Primer Mixes v4 diluted 1:4 and amplified by PCR for 24 cycles. PCR products size selection and cleanup were done using NEXTFLEX Cleanup Beads. Small exRNA libraries were eluted from beads in 15 µl and concentrations were determined using Qubit dsDNA HS Assay (Life Technologies). DNA fragment profile in small exRNA libraries was analyzed on the Bioanalyzer 2100. RNA-seq was conducted on the NovaSeq X Plus Short-Read Sequencer (Illumina, San Diego, CA) at the Genomics Research and Technology Hub, University of California Irvine.
identifiers.namespace: GEO
identifiers.id: GSM8840293_r1