GSM5215969: NT4; Homo sapiens; OTHER
Firstly, anti-CD138 MicroBeads (Miltenyi, Germany) were used to enrich plasma cells from bone marrow samples by magnetic activated cell sorting (MACS). The purity of separated plasma cells was identified by flow cytometry and was all above 90%. Total RNA was then extracted from enriched plasma cells and myeloma cells using TRIzol (Invitrogen, USA) according to the instruction manual. Prepared RNA was stored at -80 °C. RNA samples were qualified by agarose gel electrophoresis and quantified by NanoDrop ND-1000 (NanoDrop, USA). RNA integrity number (RIN) was evaluated by Agilent BioAnalyzer 2100 and was all above 7. RNA concentration and purity were also assessed (supplementary table 1). qRT-PCR was performed using ViiA 7 Real-time PCR System (Applied Biosystems) and 2 × PCR Master Mix. The expression levels of each tRFs/tiRNAs were calculated and normalized by U6 small nuclear RNA (snRNA). Total RNA samples were sequentially ligated to 3' and 5' small RNA adapters using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA). cDNA was then synthesized and amplified using Illumina's proprietary reverse transcription primers and amplification primers. Subsequently, 135-170 bp PCR amplified fragments (corresponding to 15-50 nt small RNA size range) were extracted and purified from the PAGE gel. The completed libraries were quantified by Agilent 2100 Bioanalyzer. Libraries were mixed in equal amounts according to the quantification results and used for sequencing on the instrument. And then, the DNA fragments in well-mixed libraries were denatured to generate single-stranded DNA molecules and loaded onto the reagent cartridge. Finally, the sequencing run was performed on Illumina NextSeq 500 system using NextSeq 500/550 V2 kit (Illumina) according to the manufacturer's instructions. Sequencing was carried out by running 50 cycles.