title: GSM2100162: RNASeq: 553_8; Homo sapiens; RNA-Seq
library_construction_protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency. RNA integrity was checked by Fragment Analyzer (Advanced Analytical) or 2100 Bioanalyzer using the RNA 6000 Nano assay (Agilent). All processed total RNA samples had RQN/RIN value of 6.0 or greater. The sequencing library was prepared with the standard TruSeq RNA Sample Prep Kit v2 protocol (Illumina). mRNA was isolated and fragmented. cDNA was synthesized using random hexamers, end-repaired and ligated with appropriate adaptors for sequencing. The library then underwent size selection and purification using AMPure XP beads (Beckman Coulter). The appropriate Illumina recommended 6 bp barcode bases are introduced at one end of the adaptors during the PCR amplification step. The size and concentration of the RNAseq libraries were measured by Bioanalyzer and Qubit fluorometry (Thermo Fisher Scientific) before loading onto the sequencer. The mRNA libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide single-end reads, according to the standard manufacturer's protocol (Illumina,).
attributes.tag: GEO Accession
attributes.value: GSM2100162