Next ultra rna seq library prep kit

Immediately after immunopanning, cells were scraped from each plate to harvest RNA and to minimize the delay between the initial dissociation of the hCS and RNA isolation (~3 hours). Total RNA was extracted using the miRNeasy kit (Qiagen) under the protocols of the manufacturer. The quality of the RNA was assessed by Bioanalyzer. Samples with integrity number higher than 8 were used for library construction. We used the Ovation® RNA-seq system V2 (Nugen 7102) to perform first and second-strand cDNA synthesis. We then used the Next Ultra RNA-seq library prep kit for Illumina (NEB E7530) and NEBNext® multiplex oligos for Illumina® (NEB E7335 E7500) to perform end repair, adaptor ligation, and 5–6 cycles of PCR enrichment according to manufacturer’s instructions. The quality of the libraries was assessed by bioanalyzer and qPCR and high quality libraries were sequenced by the Illumina NextSeq sequencer to obtain 75bp pair-end reads (41.0M ± 5.9M reads per sample, 80.6% ± 3.7% mapped; mean ± SD). All data has been deposited into GEO database.

Total RNA was extracted using the miRNeasy kit (Qiagen) under the protocols of the manufacturer. The quality was assessed by Bioanalyzer. Samples with RNA integrity number higher than 8 were used for library construction. We used the Ovation® RNA-seq system V2 (Nugen 7102) to perform first and second-strand cDNA synthesis. We then used the Next Ultra RNA-seq library prep kit for Illumina (NEB E7530) and NEBNext® multiplex oligos for Illumina® (NEB E7335 E7500) to perform end repair, adaptor ligation, and 5–6 cycles of PCR enrichment according to manufacturer’s instructions. The quality of the libraries was assessed by bioanalyzer and qPCR and high quality libraries were sequenced by the Illumina NextSeq sequencer to obtain 150bp pair-end reads. See detailed protocol in Supplemental Information.