title: GSM9161647: Exosomes derived from 3D cultured human umbilical cord mesenchymal stem cells; Homo sapiens; miRNA-Seq
library_construction_protocol: 1. Experimental Procedures 1.1 Sample Lysis 1.1.1 Transfer appropriate tissue sample into corresponding tissue grinding tube (tissue≤30 mg, cell≤1×107). 1.1.2 Add 600 μL Buffer RLT Plus. Homogenize the lysate for using a rotor–stator homogenizer, stay at room temperature for 5 min. 1.1.3 Centrifuge at 12,000×g for 3 min in room temperature, transfer the supernatant to AllPrep DNA spin column. 1.1.4 Centrifuge 12,000×g for 30 s in room temperature, transfer the solution from the collection tube for the RNA purification. 1.1.5 Transfer the DNA spin column to the new collection tube for DNA purification. 1.2 DNA Purification 1.2.1 Add 500μL Buffer AW1 to AllPrep DNA spin column, centrifuge at 10,000×g for 30 s and discard the filtrate. 1.2.2 Add 500μL Buffer AW2 to AllPrep DNA spin column, centrifuge at 10,000×g for 30 s and discard the filtrate. 1.2.3 Centrifuge at 16,000×g for 2 min, transfer the AllPrep DNA spin column to 1.5mL centrifuge tube. 1.2.4 Add 50μL Buffer EB to AllPrep DNA spin column, stay at room temperature for 1 min. 1.2.5 Centrifuge at 16,000×g for 2 min and collect the DNA. 1.3 RNA Purification 1.3.1 Add the equal volume of 70% ethanol to the collection tube, mix by pipetting. 1.3.2 Transfer 700 μL mixed solution, to a RNeasy spin column placed in a 2 mL collection tube. 2.3.3 Centrifuge at 10,000×g for 30 s and allow it to empty by gravity flow. 1.3.4 Add 700 μL Buffer RW1 to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.5 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.6 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.7 Centrifuge at 16,000×g for 2 min and transfer the RNeasy spin column to the 1.5 mL centrifuge tube. 1.3.8 Add 35 μL RNase-free water to RNeasy spin column. Stay at room temperature for 1min. 1.3.9 Centrifuge at 16,000×g for 2 min at room temperature to collect RNA. After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.