GSM5241740: Cancer-associated firboblasts 5; Homo sapiens; ncRNA-Seq
Cancer-associated fibroblasts (CAFs) and Primary normal fibroblasts (NFs) were isolated from pancreatic ductal carcinoma and paired non-cancerous tissues. The Human Tumor Dissociation Kit (Miltenyi Biotec 130-095-929) was used for the generation of single cells from dissociated tissues. Fibroblast populations were isolated by differential velocity adherent technique. 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).