title: GSM4119968: V255; Homo sapiens; RNA-Seq
library_construction_protocol: Exosomes were isolated from spontaneous sputum in bronchiectasis patients and induced sputum from healthy subjects. After thawing, approximately four aliquots of phosphate buffer solution were added to achieve complete homogenization, followed by 18,000 g centrifugation for 10 minutes at 4°C (Thermo Scientific, USA), which were modified by instructions, then filtered through 0.8µm filter. The supernatant was used for extraction of exosomes with the exoEasy Maxi Kit (Qiagen Inc., Hilden, Germany) based on manufacturer's instructions. Exosomes were suspended with buffer XE (Qiagen Inc., Hilden, Germany). For exosomal miRNA sequencing, the total RNA was extracted from the cell pellets by using QIAZol lysis reagent (Qiagen Inc., Hilden, Germany) by following the instructions of the manufacturer. RNA per sample as the input material for the miRNA library which was generated with NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB Inc., USA) by following the manufacturer's protocol. Briefly, NEB 3' & 5' Adaptor were directly and specifically ligated to the 3' and 5' end of the small RNAs (miRNA, siRNA and piRNA), which also termed the 'SR Adaptor'. After ligation at the 3' end, the reverse transcriptase Primer (RT Primer) was hybridized to the excess of 3' SR adaptor and transformed the single-stranded DNA adaptor into a double-strand DNA. The first strand cDNA was synthesized by using M-MuLV Reverse Transcriptase (RNase H-). PCR amplification was performed, followed by purification of the PCR products (DNA fragments, length: 140~160 bp) on a 8% polyacrylamide gel (100 V, 80 min). The cDNA library was constructed and its sequence length was examined by using the Aglient Bioanalyzer 2100 system (Aligent Technologies, CA, USA). The samples with an effective concentration being greater than 2nM as quantified with qPCR could be subject to exosomal miRNA sequencing by using Illumina HiSeq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM4119968