RNA-SN-C-PAP-rep1 (CRX2069166)
5 ul of Dynabeads MyOne Streptavidin C1 was washed with 20 ul of Dynabead solution A (50 mM NaCl and 0.1 M NaOH) twice and 20 ul of Dynabead solution B (100 mM NaCl). Dynabeads were then resuspended in 5 ul of 2x B&W buffer [10 mM Tris-HCl (pH 7.5), 2 M NaCl and 1 mM EDTA] and 5 ul of 100 uM biotinylated oligo-dT30VN (Sangon Biotech) was added for 20 min at room temperature. Dynabeads were further washed with 20 ul of 1x B&W buffer four times and resuspended in 10 ul of nuclease-free water. 25 karyoplasts or 3 cytoplasts (per sample) were lyzed in 5 ul of lysis buffer , and 0.24 ul of 5 M NaCl and 1 ul of Dynabeads were added for 20 min at room temperature. The supernatant was collected as the polyA-ve fraction, and 1 ul of 105-diluted ERCC RNA Spike-In Mix (Thermo Fisher Scientific) and 6.6 ul of nuclease-free water were added. Dynabeads were resuspended in 5 ul of lysis buffer as the polyA+ve fraction, and 1 ul of 105-diluted ERCC RNA Spike-In Mix and 7.6 ul of nuclease-free water were added. Library preparation was performed using the Ovation SoLo RNA-Seq System (Tecan) after minor modifications. The resulting libraries were purified, quantified using the Qubit 1X dsDNA HS Assay Kit and qPCR, and assessed for quality and successful amplification using a Fragment Analyzer System. Libraries were then sequenced on the NovaSeq X Plus Sequencing System.