title: CNS_RNA-seq_9C

status: Public on Jun 14 2017

city: Urbana

first: Andrew

last: Steelman

address: 1201 West Gregory

institute: University of Illinois

description: Barcode: CTGAAGCT-CAGGACGT

data_processing:
The libraries were pooled in equimolar concentration and the pool was quantitated by qPCR and sequenced on one lane for 101 cycles on a HiSeq2500 using a HiSeq SBS sequencing kit version 3.
Fastq files were generated and demultiplexed with with the bcl2fastq v1.8.4 Conversion Software (Illumina)
Each sample's fastq file run through trimmomatic 0.33 to first remove any remaining standard Illumina TruSeq SE v3 adapters, then trim bases from both ends with quality scores below 28, and finally to remove reads shorter than 30 bp (parameters ILLUMINACLIP:/home/apps/trimmomatic/trimmomatic-0.33/adapters/TruSeq3-SE.fa:2:15:10 LEADING:28 TRAILING:28 MINLEN:30)
NCBI's GRCm38.p3 genome was indexed using STAR 2.4.2a with gene models ref_GRCm38.p3_top_level.gff3.gz (NCBI Mus musculus Annotation Release 105) and parameters --sjdbOverhang 99 and --sjdbGTFtagExonParentGene gene. Each sample was then aligned to the genome again using STAR 2.4.2a with gene models ref_GRCm38.p3_top_level.gff3.gz (NCBI Mus musculus Annotation Release 105) and parameter --sjdbGTFtagExonParentGene gene.
Read counts for each gene were generated using featureCounts (from subread v 1.4.6-p4) with parameters -s 2 -g gene -t exon
Genome_build: NCBI GRCm38.p3
Supplementary_files_format_and_content: tab-delimited text files of raw read counts per gene per sample were used directly in edgeR 3.12.0 for statistical analysis and thus are the "normalized" data.

source_name: Cerebellum, Day 8

extract_protocol:
Mice were anethetized with ketamine and xylazine then perfused with sterile phosphate buffered saline (PBS).  The cerebellum and spinal cords were dissected and flash frozen in liquid nitrogen.  RNA was extracted using Tri reagent, then further purified using GeneJet RNA purification kit and DNAse digestion.
The strand-specific RNAseq libraries were prepared with Illumina's 'TruSeq Stranded RNA Sample Prep kit'. Adaptors must be remove. Sequence of adaptor for read1: AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG  (NNNNNN= 6 nt index). Sequence of adaptor for read2: AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCAT

treatment_protocol: Mice were anesthetized with isoflurane then inoculated with saline (D0) or influenza A virus (Puerto Rico/8/34; PR8, 1.0 HAU) by intranasal route.  The total innoculation volume was 0.05ml.

tag: strain

value: 8 weeks

tag: gender

value: Female

tag: infection

value: Influenza A

tag: time

value: Day 8

tag: mouse id

value: Cerebellum


title: CNS_RNA-seq_743

type: SRA

middle: J

state: IL

country: USA

description: Barcode: AGCGATAG-GGCTCTGA

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

source_name: Spinal cord, Day 8

tag: age

value: Male

tag: tissue

value: Spinal cord


title: CNS_RNA-seq_742

description: Barcode: AGCGATAG-CCTATCCT


title: CNS_RNA-seq_741

description: Barcode: AGCGATAG-TATAGCCT

source_name: Spinal cord, Day 4

value: Day 4


title: CNS_RNA-seq_740

description: Barcode: AGCGATAG-GTACTGAC


title: CNS_RNA-seq_736

description: Barcode: TCCGCGAA-CAGGACGT


title: CNS_RNA-seq_738

description: Barcode: TCCGCGAA-AGGCGAAG


title: CNS_RNA-seq_731

description: Barcode: TCTCGCGC-CCTATCCT


title: CNS_RNA-seq_733

description: Barcode: TCTCGCGC-TAATCTTA


title: CNS_RNA-seq_735

description: Barcode: TCTCGCGC-GGCTCTGA


title: CNS_RNA-seq_730

description: Barcode: TCCGCGAA-GTACTGAC


title: HIPCVAX005_ZV103_D0_PBMC

status: Public on Aug 18 2017

type: RNA

city: Atlanta

first: Thomas

last: Hagan

state: GA

address: 954 Gatewood Road

department: Emory Vaccine Center

institute: Emory University

hyb_protocol: Samples were hybridized on  HG-133-plus-PM plates

scan_protocol: Plates were scanned on GeneTitan multichannel instrument (Affymetrix)

data_processing: Probe level processing was done by RMA with log2 transform and probe summary by median polish (using the rma function in the affy package v1.44.0 in R v3.1.3).

label: biotin

organism: Homo sapiens

source_name: elderly_subject_Visit 1

label_protocol: Fragemented cRNA was lebeled with biotin

extract_protocol: RNA was isolated from whole blood Paxgene tubes using RNAeasy kit (Quagen)

tag: subject id

tag: vaccine

value: VZV

tag: cohort

value: Atlanta

tag: visit

value: Visit 1; baseline

tag: Sex

value: female

tag: age group

value: elderly

tag: race

value: white

tag: ethnicity

value: non-hispanic

tag: tissue/cell type

value: PBMC


title: HIPCVAX005_ZV101_D0_PBMC

source_name: young_subject_Visit 1

value: young


title: CNS_RNA-seq_713

description: Barcode: TCTCGCGC-GTACTGAC

value: 8 weeks

value: Non-Infected

value: Day 0


title: CNS_RNA-seq_728

description: Barcode: TCCGCGAA-TAATCTTA

value: Influenza A


title: CNS_RNA-seq_711

description: Barcode: TCTCGCGC-ATAGAGGC

source_name: Spinal cord, Day 0 (Non-Infected)

value: Female


title: CNS_RNA-seq_709

description: Barcode: AGCGATAG-AGGCGAAG


title: CNS_RNA-seq_710

description: Barcode: TCTCGCGC-TATAGCCT


title: CNS_RNA-seq_729

description: Barcode: AGCGATAG-ATAGAGGC

value: Male


title: CNS_RNA-seq_589

description: Barcode: TCTCGCGC-AGGCGAAG

value: Day 8

value: Spinal cord


title: CNS_RNA-seq_6C

description: Barcode: CTGAAGCT-TAATCTTA

source_name: Cerebellum, Day 4

value: Day 4

value: Cerebellum


title: CNS_RNA-seq_5C

description: Barcode: CTGAAGCT-AGGCGAAG


title: CNS_RNA-seq_590

description: Barcode: AGCGATAG-TAATCTTA


title: CNS_RNA-seq_585

description: Barcode: TCCGCGAA-TATAGCCT


title: CNS_RNA-seq_583

description: Barcode: TCCGCGAA-CCTATCCT


title: CNS_RNA-seq_571

description: Barcode: AGCGATAG-CAGGACGT


title: CNS_RNA-seq_4C

description: Barcode: CTGAAGCT-GGCTCTGA

source_name: Cerebellum, Day 0 (Non-Infected)


title: CNS_RNA-seq_574

description: Barcode: TCCGCGAA-GGCTCTGA


title: CNS_RNA-seq_575

description: Barcode: TCTCGCGC-CAGGACGT


title: CNS_RNA-seq_30C

description: Barcode: CGGCTATG-GTACTGAC


title: CNS_RNA-seq_3C

description: Barcode: CTGAAGCT-CCTATCCT


title: CNS_RNA-seq_29C

description: Barcode: CGGCTATG-CAGGACGT


title: CNS_RNA-seq_28C

description: Barcode: CGGCTATG-TAATCTTA


title: CNS_RNA-seq_27C

description: Barcode: CGGCTATG-AGGCGAAG


title: CNS_RNA-seq_25C

description: Barcode: CGGCTATG-CCTATCCT


title: CNS_RNA-seq_26C

description: Barcode: CGGCTATG-GGCTCTGA


title: CNS_RNA-seq_22C

description: Barcode: CGGCTATG-ATAGAGGC


title: CNS_RNA-seq_21C

description: Barcode: CGGCTATG-TATAGCCT


title: CNS_RNA-seq_1C

description: Barcode: CTGAAGCT-TATAGCCT


title: CNS_RNA-seq_20C

description: Barcode: TAATGCGC-GTACTGAC


title: CNS_RNA-seq_13C

description: Barcode: TAATGCGC-CCTATCCT


title: CNS_RNA-seq_17C

description: Barcode: TAATGCGC-AGGCGAAG


title: CNS_RNA-seq_14C

description: Barcode: TAATGCGC-GGCTCTGA


title: CNS_RNA-seq_10C

description: Barcode: CTGAAGCT-GTACTGAC


title: CNS_RNA-seq_2C

description: Barcode: CTGAAGCT-ATAGAGGC


title: CNS_RNA-seq_12C

description: Barcode: TAATGCGC-ATAGAGGC


title: CNS_RNA-seq_11C

description: Barcode: TAATGCGC-TATAGCCT


title: RNASeq: 952_3

status: Public on Dec 28 2016

city: Stanford

first: Joshua

last: Knowles

state: California

address: 300 Pasteur Dr.

institute: Stanford University

description:
RNASeq data from hiPSC line 952_3 from individual 952
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.

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

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: SD

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

tag: passage

tag: state

value: Insulin sensitive

value: White

tag: sspg

tag: bmi

tag: indiv


title: RNASeq: 987_1

description:
RNASeq data from hiPSC line 987_1 from individual 987
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: PEC

value: Insulin resistant

tag: age

tag: Sex

tag: race


title: RNASeq: 963_1

description:
RNASeq data from hiPSC line 963_1 from individual 963
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: batch

tag: reprogramming source cell

tag: reprogramming batch


title: RNASeq: 963_5

description:
RNASeq data from hiPSC line 963_5 from individual 963
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: sendai virus lot

tag: reprogrammed by

tag: rna prep by


title: RNASeq: 998_5

description:
RNASeq data from hiPSC line 998_5 from individual 998
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: Activated T-Cells

tag: rna method

tag: matrigel dilution

tag: rin


title: RNASeq: 998_2

description:
RNASeq data from hiPSC line 998_2 from individual 998
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: rna harvest date

tag: passage


title: RNASeq: 963_3

description:
RNASeq data from hiPSC line 963_3 from individual 963
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: state

tag: sspg


title: RNASeq: 987_3

description:
RNASeq data from hiPSC line 987_3 from individual 987
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: age

tag: bmi

tag: indiv


title: RNASeq: 961_3

description:
RNASeq data from hiPSC line 961_3 from individual 961
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: Sex

tag: race


title: RNASeq: 998_1

description:
RNASeq data from hiPSC line 998_1 from individual 998
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: reprogramming source cell

tag: reprogramming batch

tag: sendai virus lot


title: RNASeq: 987_2

description:
RNASeq data from hiPSC line 987_2 from individual 987
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: 928_6

description:
RNASeq data from hiPSC line 928_6 from individual 928
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: 928_4

description:
RNASeq data from hiPSC line 928_4 from individual 928
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: 952_5

description:
RNASeq data from hiPSC line 952_5 from individual 952
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: sspg


title: RNASeq: 998_4

description:
RNASeq data from hiPSC line 998_4 from individual 998
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: bmi

tag: indiv


title: RNASeq: 998_6

description:
RNASeq data from hiPSC line 998_6 from individual 998
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: Sex

tag: race


title: RNASeq: 961_1

description:
RNASeq data from hiPSC line 961_1 from individual 961
Peripheral Mononuclear Cell Isolation: After an overnight fast, blood was drawn in cell preparation tubes (BD Vacutainer CPT tube) with sodium citrate for the isolation of peripheral blood mononuclear cells. The tubes were centrifuged at 1800 g for 40 minutes at room temperature.  The plasma and cells in the interphase were transferred to a new tube, resuspended in RPMI medium and centrifuged at 1500 rpm for 20 minutes at room temperature. The cells were frozen in RPMI medium supplemented with 12.5% of human serum albumin and 10% of DMSO and stored in liquid nitrogen for later use.
Recruitment: The study included 201 subjects who had volunteered between October 2002 and October 2013 and were in general good health. Stanford Institutional Review Board approved the study protocol, and all subjects gave written informed consent for study participation.  The study was performed at the Stanford Clinical and Translational Research Unit. Height and weight were measured while subjects were wearing light clothing and no shoes. Body mass index was calculated by dividing weight (in kilograms) by the square of height (in meters).
Reprogramming: T-cells: PBMCs were thawed into 6-well plates pre-coated with hCD3 antibody (Ebioscience) and grown in LGM media (Lonza Clonetics) supplemented with 50 ng/ml IL-2 (R&D Systems), 0.4 μg/ml CD3 (Thermo Fisher Scientific) and CD28 (Thermo Fischer Scientific), 1 mM L-Glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific) for 6 days until approximately 80% of cells express CD3 and CD28. Activated T-cells (5.0 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune 1.0 kit (Thermo Fisher Scientific) at a multiplicity of infection (MOI) 10 for all factors, and grown in LGM media supplemented with IL-2, CD3, L-Glutamine, and Penicillin-Streptomycin. After 24 hours, transduced cells were plated onto 0.1% Gelatin/PBS coated plates containing a monolayer of mouse embryonic fibroblasts (MEF)-irradiated cells (GlobalStem) and grown in iPSC A medium containing DMEM, 15% Fetal Calf Serum (FCS) (Gemini Biologicals), 1 mM L-Glutamine, 1% Penicillin-Streptomycin for 4 days. The cells were switched to iPSC B medium containing DMEM/F12, 20% Knockout Serum (Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Thermo Fisher Scientific), 1mM L-Glutamine, 1% Penicillin-Streptomycin, 1 μg/mL Fungizone (Hyclone), 2-mercaptoethanol (Sigma), 20 ng/ml bFGF (R&D Systems), and 50 μg/mL Ascorbic Acid (Sigma) for the duration of reprogramming.
Reprogramming: Erythroblast protocol: PBMCs were thawed into 6-well plates and expanded as erythroblasts in Expansion medium (EM) containing QBSF-60 (Quality Biologicals), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (Amgen) 40 ng/ml IGF-1 (R&D Systems), 1 μM Dexamethasone (Sigma), 1% Penicillin-Streptomycin, 50 μg/ml Ascorbic Acid (Sigma), 1 μg/mL Fungizone for 9-12 days until approximately 90% of cells expressed CD36 and CD71.  Erythroblasts (1.5-2.25 E+05 cells) were transduced with the four Sendai viruses expressing Oct3/4, Sox2, Klf4, and c-Myc using the Cytotune1.0 kit at an MOI of 10 for all factors. After 24 hours, transduced cells were washed and plated onto 0.1% Gelatin/PBS coated plates containing a monolayer MEF cells and grown in iPSC A medium containing DMEM/F12 10% FCS, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin , 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 3 days. Cells were switched to medium containing 50% IPSC A medium and 50% iPSC Medium B containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 ug/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF, and 50 μg/mL Ascorbic Acid for 2 days and switched to 100% IPSC B medium for the duration of reprogramming. Primary iPSC colonies emerged at day 9-12 post-transduction. To sub-clone passage 1 (P1) colonies, iPSC cultures were subjected to live cell immunostaining with DAPI (Thermo Fisher Scientific) and anti-Human Tra-1-60 PE (Ebioscience) to identify pluripotent colonies. Tra-1-60+ colonies were manually picked using a fluorescent microscope housed in a biosafety cabinet and transferred with a micropipette to a 12-well plate coated with 0.1% Gelatin/PBS containing MEFs. iPSC P1 colonies were maintained in hESC medium containing DMEM/F12, 20% Knockout Serum, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 1mM Penicillin-Streptomycin, 1 μg/mL Fungizone, 2-mercaptoethanol, 10 ng/ml bFGF.  IPSC cultures were routinely immunostained with human anti-Oct4 (Stemgent) and anti-Nanog (R&D Systems) to assess pluripotency.

tag: reprogramming batch

tag: sendai virus lot

tag: reprogrammed by