GSM8635539: enriched nuclei_HC_1489; Homo sapiens; OTHER
RNA extractions from sorted nuclei were carried out with FFPE RNA Purification kits (Norgen: Product # 25300) using the manufacturer's protocol from Step 2 (Lysate Preparation) with the following modifications: The On-column DNA removal Protocol was omitted. RNA binding and Column Wash centrifuge steps were carried out at 1,789×g for 2 min, followed by 1,789×g for 5 min to dry the resin. Elution was carried out in two steps, with 25 µL Elution Solution A added to each column followed by a 1 min room temperature incubation and centrifugation at 200×g for 2 min, then a further 15 µL Elution Solution A was added to each column with centrifugation at 1,789×g for 5 min. For samples containing >2,000 nuclei, the final 30 µL elution was split into two 15 µL aliquots. Purified RNA was vacuum dried in a CentriVap Benchtop Vacuum Concentrator (Labconco) for 40 min (or until no liquid was visible) at 4˚C and either used directly for library preparation or stored at -20˚C. Sequencing library generation was carried out using the Trio RNA-Seq, Human rRNA Library Preparation Kit (Tecan: Product # 0506-A01) using the manufacturer's protocol with the following modifications: Lyophilized RNA was resuspended in 12.5 µL DNAse mix and incubated at 37˚C for 30 min followed by 60˚C for 5 min and 4˚C Hold. Library Amplification I was carried out as 72˚C 2 min/95˚C 2 min/2×(95˚C 30 s/ 60˚C 60 s)/6×(95˚C 30 s/ 65˚C 60 s)/65˚C 5 min/4˚C Hold Library Amplification II was carried out as 95˚C 2 min/2×(95˚C 30 s/ 60˚C 60 s)/6×(95˚C 30 s/ 65˚C 60 s)/65˚C 5 min/4˚C Hold Final Libraries eluted in 30 µL DNA Resuspension Buffer (DR1) were checked on Agilent Bioanalyzer DNA 1000 chips, then measured on a Nanodrop 8000 and 32 sample pools (normalized to 500 ng/sample then diluted to 5 ng/µL) were sent for sequencing on an Illumina NextSeq High Output platform.