title: RNASeq: 110_3

description:
RNASeq data from hiPSC line 110_3 from individual 110
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rna prep by

tag: rna method

tag: rna prep date


title: RNASeq: 110_1

status: Public on Dec 28 2016

type: SRA

city: Stanford

first: Joshua

last: Knowles

state: California

address: 300 Pasteur Dr.

country: USA

institute: Stanford University

description:
RNASeq data from hiPSC line 110_1 from individual 110
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

library_source: transcriptomic

data_processing:
RNAseq reads were aligned to GRCh37 with STAR v2.4.0g1 (Dobin et al., 2013).  Uniquely mapping reads overlapping genes were counted with featureCounts v1.4.4 (Liao et al., 2014) using annotations from ENSEMBL v70.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RNA-seq count  values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: human induced pluripotent stem cells

growth_protocol: iPSCs were expanded on 12-well plates coated with 0.1% Gelatin/PBS containing MEFs until passage 6-8 and were maintained in hESC medium as described. iPSCs were bulk passaged using either Accutase (Innovative Cell technologies) or 1 mM EDTA (Sigma). Cells were washed once with PBS and treated with pre-warmed Accutase or EDTA, incubated at 37 degrees for 1-5 minutes, washed once with PBS for Accutase passaging, and resuspended in fresh hESC medium containing either 10 μM Y-27632 Rock inhibitor (RI) (Stemgent) or 2 μM Thiazovivin (Millipore). Cells were detached with gentle pipetting and cell suspension was transferred to a new 12-well plate coated with 0.1% Gelatin/PBS containing a MEFs. After 12 hours incubation with RI or Thiazovivin, medium was changed daily with fresh hESC medium. iPSCs were passaged every 5-7 days and transitioned from MEF-feeder to feeder-free conditions at passage 6-8. To transition to feeder-free conditions, iPSCs were passaged as described and transferred to a 12-well or 6-well plate coated with 5% Matrigel (BD Bioscience) in DMEM/F-12. Cells were maintained in mTesr1 (Stem Cell Technologies) supplemented with 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 0.1 μg/ml Fungizone.  To ensure Sendai virus was cleared from cells, it was tested by immunostaining cultures with anti-Sendai virus antibody (Abcam). Differentiating colonies were routinely eliminated from cultures.

extract_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,).

tag: batch

tag: reprogramming source cell

value: Erythroblasts

tag: reprogramming batch

tag: sendai virus lot

tag: reprogrammed by

value: PEC

value: YL

value: Life-Tech GITC

tag: matrigel dilution

tag: rin

tag: rna harvest date

tag: passage

tag: state

value: Insulin sensitive

tag: age

tag: Sex

value: Male

tag: race

value: White

tag: sspg

tag: bmi

tag: indiv


title: RNASeq: 107_4

description:
RNASeq data from hiPSC line 107_4 from individual 107
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rna prep by

tag: rna method

tag: rna prep date

value: Female


title: RNASeq: 102_4

description:
RNASeq data from hiPSC line 102_4 from individual 102
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: batch

tag: reprogramming source cell

tag: reprogramming batch

value: AP

value: African American


title: RNASeq: 102_6

description:
RNASeq data from hiPSC line 102_6 from individual 102
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: sendai virus lot

tag: reprogrammed by

tag: matrigel dilution


title: RNASeq: 107_5

description:
RNASeq data from hiPSC line 107_5 from individual 107
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rin

tag: rna harvest date

tag: passage


title: RNASeq: 081_5

description:
RNASeq data from hiPSC line 081_5 from individual 081
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: state

value: Insulin resistant

tag: age

tag: Sex


title: RNASeq: 081_4

description:
RNASeq data from hiPSC line 081_4 from individual 081
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: race

tag: sspg

tag: bmi


title: RNASeq: 081_3

description:
RNASeq data from hiPSC line 081_3 from individual 081
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rna prep by

tag: rna method

tag: indiv


title: RNASeq: 134_3

description:
RNASeq data from hiPSC line 134_3 from individual 134
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: batch

tag: reprogramming source cell

value: Asian


title: RNASeq: 072_3

description:
RNASeq data from hiPSC line 072_3 from individual 072
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: reprogramming batch

tag: sendai virus lot

value: SD

tag: rna prep date


title: RNASeq: 072_2

description:
RNASeq data from hiPSC line 072_2 from individual 072
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: reprogrammed by

tag: matrigel dilution

tag: rin


title: RNASeq: 072_4

description:
RNASeq data from hiPSC line 072_4 from individual 072
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rna harvest date

tag: passage

tag: state


title: RNASeq: 071_3

description:
RNASeq data from hiPSC line 071_3 from individual 071
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: age

tag: Sex

tag: race


title: RNASeq: 071_4

description:
RNASeq data from hiPSC line 071_4 from individual 071
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rna prep by

tag: sspg

tag: bmi


title: RNASeq: 060_7

description:
RNASeq data from hiPSC line 060_7 from individual 060
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: batch

tag: rna method

tag: indiv


title: RNASeq: 071_2

description:
RNASeq data from hiPSC line 071_2 from individual 071
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: reprogramming source cell

tag: reprogramming batch

tag: sendai virus lot


title: RNASeq: 060_3

description:
RNASeq data from hiPSC line 060_3 from individual 060
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: reprogrammed by

tag: matrigel dilution


title: RNASeq: 060_4

description:
RNASeq data from hiPSC line 060_4 from individual 060
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast 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.

tag: rin

tag: rna harvest date