GSM7026699: Day 10 of hFOB differentiation replicate 1 [t10a]; Homo sapiens; RNA-Seq
RNA was extracted from 3 biological replicates of hFOB cells at days (0,2,4, and 10) using Qiagen RNeasy mini kit. Extracted RNA was  used to create full length cDNA using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification kit (New England Biolabs Ipswich MA Lot#10078130). Following the PacBio Isoseq protocol, first and second strand cDNA were synthesized using NEB dT oligo and the PacBio Template Switching Oligo. For the cDNA amplification 14 cycles of PCR were performed followed by a Pronex bead cleanup (Promega Corporation Madison WI, Lot #NG103A). The amplified and purified cDNA were QCed using the Bioanalyzer DNA 12000 kit Approximately 300 ng of cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced libraries using 11 SMRT cells on the Sequel II system using polymerase v2.1 with a loading concentration of 85pM. A 2-h extension and 30-h movie collection time was used for data collection. The “ccs” command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads (~22  million) into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.