GSM8253420: Evs-PANC-1-rep3; Homo sapiens; ncRNA-Seq
PANC-1 cells were treated with CAF-derived EVs for 2 weeks and then collected for RNA extraction After the quality inspection of Agilent 2100 Bioanalyzer (Agilent, cat.G2939AA, CA, USA) and NanoPhotometer® (Implen, cat.N60, Munich, Germany), ribosomal RNA was removed from 1 μg total RNA using Ribo-off® rRNA Depletion Kit (Human/Mouse/Rat, Vazyme, cat.N406-01, Nanjing, China) and purified using VAHTS® RNA Clean Beads (Vazyme, cat. N412, Nanjing, China). VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, cat.NR604-02, Nanjing, China) was used for lncRNA library construction following the manufacturer's protocol. The enriched ncRNAs were segmented with FRAG/PRIME Buffer. The first cDNA strand was generated by reverse transcription starting from -3 ' , and the second cDNA strand with U was synthesized using the first cDNA strand. Single- or dual-index adapters were ligated to the fragments with ligase, and size selection was performed with VAHTS® DNA Clean Beads (Vazyme, cat.N411, Nanjing, China). The ligated products were amplified with PCR by the following conditions: Incubated at 37 ° C for 10 minutes to remove the U containing antisense chain, initial denaturation at 98℃ for 10 sec; 12-14 cycles of denaturation at 98℃ for 10 sec, annealing at 60℃ for 30 sec, extension at 72℃ for 30 sec, and then final extension at 72 ℃ for 5 min. The average insert size for the final cDNA library was 290bp (±50 bp). At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (Illumina Corporation， San Diego，USA) following the vendor's recommended protocol by Guangzhou Huayin Health Medical Group CO.,Ltd. (Guangzhou, China).