title: RNASeq: 060_1

description:
RNASeq data from hiPSC line 060_1 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: passage

tag: state


title: RNASeq: 107_3

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 107_3 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.

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

tag: rna prep by

value: YL

tag: rna method

value: Life-Tech GITC

tag: matrigel dilution

tag: rin

tag: rna harvest date

tag: rna prep date

value: Insulin sensitive

tag: age

tag: Sex

value: Female

tag: race

value: White

tag: sspg

tag: bmi

tag: indiv


title: RNASeq: 060_2

description:
RNASeq data from hiPSC line 060_2 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: passage

tag: state

value: Insulin resistant


title: RNASeq: 054_2

description:
RNASeq data from hiPSC line 054_2 from individual 054
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

value: AP

value: Asian


title: RNASeq: 054_1

description:
RNASeq data from hiPSC line 054_1 from individual 054
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: rna prep by

tag: rna method


title: RNASeq: 054_3

description:
RNASeq data from hiPSC line 054_3 from individual 054
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: matrigel dilution

tag: rin

tag: rna harvest date


title: RNASeq: 023_3

description:
RNASeq data from hiPSC line 023_3 from individual 023
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 date

tag: age

tag: Sex

value: Male


title: RNASeq: 024_5

description:
RNASeq data from hiPSC line 024_5 from individual 024
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: 023_2

description:
RNASeq data from hiPSC line 023_2 from individual 023
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: passage

tag: indiv


title: STL001_Spleen Rep1

status: Public on Nov 15 2016

city: La Jolla

first: Anthony

middle: D

last: Schmitt

address: 9500 GILMAN DR., BLDG. CMME RM 2071

department: Cellular and Molecular Medicine

institute: UC San Diego

description: STL001_Spleen Rep1

library_source: genomic

data_processing:
Library strategy: HiC-Seq
Each Hi-C read end was mapped indepenedently using BWA -mem and default parameters. BWA version 0.7.8 was used.
Read ends with >1 alighment were filtered and only 5' alignments were kept using in-house Python script.
SAM files were converted to BAM files and sorted using Samtools Version 0.1.18
PCR duplicates were removed using PicardTools Version 1.49
Reads aligning >500bp from Hi-C restriction enzyme cutsites were removed and reads <15kb insert size were removed using in-house scripts.
Raw contact matrices were constructed using in-house scripts, or normalization was applied using HiCNorm, VC, or ICE, where noted.
FIREs were identified by first applying HiCNormCis on the raw contact marix, as described in this manuscript.
TAD boundaires were identified by applying the insulation square method on the HiCNorm matrices, as previously described (Crane et al., 2015)
A/B compartments were identified as previously described (Lieberman-Aiden et al., 2009)
Statistically significant Hi-C contacts were identified using Fit-Hi-C, as previously described (Ay et al., 2014)
Genome_build: Human data is in hg19, mouse data is in mm9.
Supplementary_files_format_and_content: Hi-C matrices are provided at 40kb bin resolution in NxN format for each chromosome for either raw contact maps, or Hi-C matrices normalized with HiCNorm, or HiCNorm followed by quantile normalization using samples from the same published or unpublished source. Even if a dataset was not specifically used in this study, a contact matrix was generated for each sample, for each study. For example, all samples from Zuin et al, 2014 are quantile normalized together.  FIRE and TAD boundary annotations are provided as a unique BED file for each sample. Compartment A/B annotations are provided as a unique bigwig file for each sample. Fit-Hi-C output is provided in a 7-column format, corresponding to position 1, position 2, observed count, expected count, O/E, p-value, and q-value. Only the upper-triangle matrix entries (where i<=j)  are provided, and only pairwise contacts within 2Mb genomic distance are provided.

molecule: genomic DNA

source_name: Spleen Tissue

extract_protocol:
For newly generated Hi-C datasets presented in this study, Hi-C was performed as previously described (Lieberman-Aiden et al., 2009) with minor modifications pertaining to the handling of flash-frozen human tissue as previously described (Leung et al., 2015). Flash frozen human tissues were first pulverized using mortar as pestle, and then crosslinked for 25min using 1% formaldehyde, quenched with Glycine, washed with PBS and then input into the remaining Hi-C protocol or flash frozen for future analysis. For each experiment, ~65mg of tissue was used for subsequent Hi-C library preparation, but varied from tissue to tissue depending on cellular density of each tissue. Using ~65mg of tissue, samples were lysed and dounced to obtain single nuclei suspensions. Chromatin was then solubilized with SDS, and then quenched with TritonX-100. Chromatin was digested overnight with HindIII. Digested ends were filled-in dNTPs, except a biotinylated dCTP was used to label digested ends. Nuclei were then lysed using SDS, and chromatin complexes were diluted. Ligation was performed in large volumes to favor intra-molecular ligation events. Crosslinks were reversed by overnight incubation with Proteinase K and high temperatures. DNA was then purified using two rounds of Pheno-Chloroform purification, followed by ethanol precipitation. Biotinylated nucleotides on unligated ends were removed by a T4 DNA polyermase reaction, and purified using ethanol precipitation. DNA was then quantified using Qubit Fluorometer, and <=5ug of DNA was used for subsequent library preparation.
Libraries were constructed using a modifed Illumina TruSeq library preparation method specific to the preparation of Hi-C libraries for Illumina sequencing, as previously described (Lieberman-Aiden et al, 2009). First Hi-C ligation products were sheared to ~400bp fragment size. Sheard ends were repaired using a combination of T4 PNK, T4 DNA polymerase, and Klenow. The reaction was purified using Qiagen MinElute columns. DNA molecules were then dA-tailed using Klenow(exo-) and then size selected from 200-600bp using agaorse gel and purified. Biotin-labeled DNA species were then enriched using streptavidin-coated beads and Illumina TruSeq adapters were ligated using T4 DNA ligase while DNA is bound to the streptavidin beads. Beads were then washed, and PCR amplified to obtain enough material for Illumina sequencing.

tag: tissue

value: Spleen Tissue


title: RNASeq: 024_1

description:
RNASeq data from hiPSC line 024_1 from individual 024
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

tag: state


title: RNASeq: 018_3

description:
RNASeq data from hiPSC line 018_3 from individual 018
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: rna prep by

tag: rna method

tag: matrigel dilution


title: RNASeq: 023_5

description:
RNASeq data from hiPSC line 023_5 from individual 023
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: rna prep date

tag: age


title: RNASeq: 018_4

description:
RNASeq data from hiPSC line 018_4 from individual 018
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: Sex

tag: race

tag: sspg

tag: bmi


title: STL002_SmallBowel Rep1

description: STL002_SmallBowel Rep1

source_name: Small Intenstine Tissue

tag: tissue

value: Small Intenstine Tissue


title: STL003_Spleen Rep1

description: STL003_Spleen Rep1


title: STL003_RightVentricle Rep1

description: STL003_RightVentricle Rep1

source_name: Right Ventricle Tissue

value: Right Ventricle Tissue


title: STL001_Psoas Rep2

description: STL001_Psoas Rep2

source_name: Psoas Muscle Tissue

value: Psoas Muscle Tissue


title: STL001_Psoas Rep1

description: STL001_Psoas Rep1


title: STL003_Pancreas Rep3

description: STL003_Pancreas Rep3

source_name: Pancreas Tissue

value: Pancreas Tissue


title: STL003_Pancreas Rep2

description: STL003_Pancreas Rep2


title: STL003_Pancreas Rep1

description: STL003_Pancreas Rep1


title: STL002_Pancreas Rep1

description: STL002_Pancreas Rep1


title: STL003_Psoas Rep1

description: STL003_Psoas Rep1


title: RNASeq: 120_4

description:
RNASeq data from hiPSC line 120_4 from individual 120
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: passage

value: Hispanic

tag: indiv


title: STL002_Ovary Rep1

description: STL002_Ovary Rep1

source_name: Ovary Tissue

value: Ovary Tissue


title: STL002_Lung Rep1

description: STL002_Lung Rep1

source_name: Lung Tissue

value: Lung Tissue


title: Dorsolateral Prefrontal Cortex

description: Dorsolateral Prefrontal Cortex

source_name: Cortex Tissue

value: Cortex Tissue


title: STL001_Lung Rep1

description: STL001_Lung Rep1


title: RNASeq: 024_4

description:
RNASeq data from hiPSC line 024_4 from individual 024
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.

value: Erythroblasts

tag: reprogramming batch

tag: sendai virus lot

tag: reprogrammed by

value: PEC

value: YL

value: Life-Tech GITC

tag: state


title: Hippocampus

description: Hippocampus

source_name: Hippocampus Tissue

value: Hippocampus Tissue


title: GZ, individual 3

status: Public on Oct 20 2016

city: Chapel Hill

first: Hyejung

last: Won

state: North Carolina

address: 116 Manning Dr.

department: Genetics

institute: UNC

description:
processed data file: FBP_IC-heatmap-chr-10k.hdf5
processed data file: FBP_IC-heatmap-chr-40k.hdf5
processed data file: FBP_IC-heatmap-res-100k.hdf5
processed data file: Sczloci_interacting_region_FBP_FDR.bed
processed data file: Evolloci_interacting_region_enhctrl_FBP_FDR.bed

data_processing:
Library strategy: Hi-C
alignment: bowtie2 (--score-min -L 0.6,0.2--very-sensitive)
filtering and bias correction: hiclib
normalization: hiclib
Genome_build: hg19
Supplementary_files_format_and_content: *hdf5 files contain a normalized chromatin contact matrix at different resolutions, included in file name (10kb, 40kb, 100kb); Sczloci*.bed files contain schizophrenia GWAS loci interacting regions, and Evolloci*.bed files contain Human-gained enhancer interacting regions; CP_TAD.bed file contains TAD coordinates in bed file format for CP (cortical plate); GZ_TAD.bed contains TAD coordinates in bed file format for GZ (germinal zone).

source_name: Fetal brain

extract_protocol:
Fetal brains dissected into cortical plates (CP) and germinal zone (GZ).
Tissue was fixed in 1% formaldehyde for 10 min. Cross-linked DNA was digested by restriction enzyme HindIII. Digested chromatin ends were filled and marked with biotin-14-dCTP. Resulting blunt-end fragments were ligated under dilute concentration to minimize random intermolecular ligations. DNA purified after crosslinking was reversed by proteinase K treatment. Biotins from unligated ends were removed by exonuclease activity of T4 DNA polymerase. DNA was sheared by sonication and 300-600bp fragments were selected. Biotin-tagged DNA, which is intermolecular ligation products, was pulled down with streptavidin beads (Invitrogen, 65001), and ligated with Illumina paired end adapters. Resulting Hi-C library was amplified by PCR and sequenced by Illumina 50bp paired-end sequencing.

tag: developmental stage

value: Postconception week 17

value: Fetal cortex

tag: layers

value: Germinal zone


title: STL001_Bladder Rep2

description: STL001_Bladder Rep2

source_name: Bladder Tissue

value: Bladder Tissue


title: STL001_Bladder Rep1

description: STL001_Bladder Rep1


title: GZ, individual 1

value: Postconception week 18


title: STL002_Adrenal Rep1

description: STL002_Adrenal Rep1

source_name: Adrenal Tissue

value: Adrenal Tissue


title: GZ, individual 2


title: CP, individual 3

description:
processed data file: FBD_IC-heatmap-chr-10k.hdf5
processed data file: FBD_IC-heatmap-chr-40k.hdf5
processed data file: FBD_IC-heatmap-res-100k.hdf5
processed data file: Sczloci_interacting_region_FBD_FDR.bed
processed data file: Evolloci_interacting_region_enhctrl_FBD_FDR.bed

value: Cortical plate


title: CP, individual 2


title: RNASeq: 018_2

description:
RNASeq data from hiPSC line 018_2 from individual 018
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: matrigel dilution

tag: rin

tag: rna harvest date

tag: rna prep date

value: Insulin resistant

value: Male

value: White


title: CP, individual 1

tag: tissue


title: SD16

status: Public on Aug 01 2016

type: RNA

city: Boston

first: Romy

middle: B

last: Christmann

state: MASSACHUSETTS

address: 715 Albany Street E5

institute: Boston University School of Medicine

hyb_protocol: Not provided

scan_protocol: The signals of the samples were amplified and microarrays were immediately scanned using Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix Santa Clara, CA).

data_processing: The resulting CEL files were summarized using Affymetrix Expression Console (current version 1.1). The MAS5 algorithm with global scaling normalization was used to generate gene-level expression data.

label: biotin

source_name: SD16 middle lobe

label_protocol: Biotin-labeled amplified total RNA was purified, fragmented, and hybridized to Affymetrix GenCHipmiRNA 3.0 microarrays.

extract_protocol: microRNA was isolated using microRNeasy Mini Kits Qiagen protocol

tag: patient reference

value: SSc-I

value: lung

tag: diagnosis

value: Systemic Sclerosis with interstitial lung disease (SSc-ILD)


title: SD03

source_name: SD03 lower lobe

value: SSc-G


title: SD11

source_name: SD11 middle lobe

value: SSc-H


title: HC2

source_name: healthy lung

value: healthy (control)


title: SD17

source_name: SD17 lower lobe

value: SSc-J


title: HC3


title: HC5


title: HC4


title: SD22

source_name: SD22 middle lobe

value: SSc-E


title: SD08

source_name: SD08 middle lobe

value: SSc-D


title: SD02

source_name: SD02 lower lobe

value: SSc-F


title: SD05

source_name: SD05 middle lobe

value: SSc-B


title: SD28

source_name: SD28 middle lobe

value: SSc-C


title: SD14

source_name: SD14 lower lobe


title: SD07

source_name: SD07 lower lobe


title: SD31

source_name: SD31 lower lobe


title: SD23

source_name: SD23 middle lobe

value: SSc-A


title: SD13

source_name: SD13 lower lobe


title: GRO-seq_LucKD_Veh_MCF-7_2

status: Public on Jun 09 2016

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

description: nascent RNA

data_processing:
Library strategy: GRO-seq
Trimmed human GRO-seq reads were aligned to the human reference genome (hg19) using the bwa aligner {Li and Durbin, 2009} with default settings (uniquely aligned, 2 mismatches allowed, and 19 bp seed sequence).  The 5’-most base pair from each read was used in all analyses, with no more than 2 duplicates allowed at any genomic location.
Genome_build: hg19
Supplementary_files_format_and_content: bed

source_name: GRO-seq in vehicle treated MCF7 cells with shRNA-mediated knockdown of Luciferase as a control

growth_protocol: MCF-7 cells were maintained in Minimum Essential Medium Eagle supplemented with 5% calf serum containing 0.5 μg/mL puromycin and 800 μg/mL G418.

extract_protocol:
MCF-7 cells were washed three times with ice-cold PBS and then resuspended for swelling in ice-cold Hypotonic Lysis Buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, and 1 mM DTT containing 1x protease inhibitor cocktail (Sigma-Aldrich) and 4 units/mL SUPERase-In (Ambion)].   The swollen cells were collected by centrifugation at 1000 RCF for 10 min at 4°C and then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening tip 30 to 50 times to lyse the cells and release the nuclei.  The nuclei were collected by centrifugation and washed once with 1 mL of Hypotonic Lysis Buffer.  After a final collection by centrifugation, the resulting pellets of nuclei were resuspended in 500 μL of Freezing Buffer (50 mM Tris•HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 4 units/mL of SUPERase-In per mL), counted, frozen in liquid nitrogen in 100 μL aliquots containing 5 x 106 nuclei, and stored at -80°C until use.
Nuclear run-on and GRO-seq library preparation were performed as previously described {Core, 2008;Hah, 2011}.  Briefly, nuclear run-on reactions were performed for ~100 bases in the presence of sarksoyl (to prevent reengagement of RNA polymerases), rNTPs, α32P-CTP, and 5-bromo-UTP.  The nascent RNAs were isolated, hydrolyzed to ~100 bases, and enriched using α-BrdUTP antibody-conjugated agarose beads (Santa Cruz).  The bound RNAs were washed several times and eluted.  The 5’ RNA cap was removed and the ends were repaired in preparation for adapter ligation.  Small RNA adapters were ligated to the 5’ end, followed by another bead binding enrichment using α-BrdUTP antibody-conjugated agarose beads.  These steps were repeated using a 3’ adapter.  The resulting RNAs were reverse transcribed, amplified using PCR, and analyzed by high throughput sequencing using an Illumina 1G Genome Analyzer.

treatment_protocol: Prior to all experiments, MCF-7 cells were grown for 3 days in phenol red-free MEM Eagle medium supplemented with 5% charcoal-dextran-treated calf serum and then treated with either vehicle (ethanol) or 100 nM estradiol for 40 minutes.

tag: cell line

tag: cell type

value: human breast cancer cells

tag: gender

value: female


title: SD26

source_name: SD26 lower lobe

value: SSc-K


title: HC1


title: GRO-seq_PARP-1KD_Veh_MCF7_2

description: nascent RNA

source_name: GRO-seq in vehicle treated MCF7 cells with shRNA-mediated knockdown of PARP-1


title: GRO-seq_PARP-1KD_Veh_MCF7_1

description: nascent RNA


title: GRO-seq_LucKD_Veh_MCF-7_1

description: nascent RNA