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Sample GSM2991886

Query DataSets for GSM2991886

Status

Public on Aug 08, 2018

Title

14_CM_1_FC1

Sample type

SRA

Source name

induced pluripotent stem cells

Organism

Pan troglodytes

Characteristics

cell line: 3624
Sex: Male
population: Chimpanzee
cell type: induced pluripotent stem cells

Treatment protocol

Cardiomyocytes were generated following a recently protocol ((Rana et al. 2012; Burridge et al. 2014; Burridge et al. 2015)) with minimal modification. At 12 hours prior to initiating differentiation, iPSC lines at 70-90% confluence were seeded to achieve a starting density of 150,000-250,000 cells/cm2. Differentiations were initiated by removing stem cell maintenance media and adding RPMI base media (with HEPES LifeTechnologies# 22400105) supplemented with 6 μM CHIR9902 (LClabs# C-6556), 0.5 mg/mL BSA (Sigma-Aldrich# A2153), 0.213 mg/mL ascorbic acid (Santa Cruz Bio# sc-228390), 0.3 μg/mL sodium selenite (Sigma-Aldrich# S5261), 5 μg/mL Holo-transferrin (Sigma-Aldrich# T3705), 1 μg/mL Linolenic acid (Sigma-Aldrich# L2376), 1 μg/mL Linoleic acid (Sigma-Aldrich# 1012) and 1x Pen/Strep (Days 0-1 media). After 48 hours the media was changed to RPMI base media (with HEPES LifeTechnologies# 22400105) supplemented with 2 μM Wnt-c59 (Tocris# 5148), 0.5 mg/mL BSA (Sigma-Aldrich# A2153), 0.213 mg/mL ascorbic acid (Santa Cruz Bio# sc-228390 and 1x Pen/Strep (Days 2-3 media). After another 48 hours, the media was changed to RPMI base (with HEPES LifeTechnologies# 22400105) supplemented 0.5 mg/mL BSA (Sigma-Aldrich# A2153), 0.213 mg/mL ascorbic acid (Santa Cruz Bio# sc-228390) and 1x Pen/Strep. Media was changed every 48 hours until day 10 (total of 2 additional media changes). At day 10 the media was switched to RPMI media (no glucose, Cellgro#10-043-CV) supplemented with 5 mM Sodium D/L Lactate (Sigma-Aldrich# L4263), 0.5 mg/mL BSA (Sigma-Aldrich# A2153), 0.213 mg/mL ascorbic acid (Santa Cruz Bio# sc-228390) and 1x Pen/Strep, media was changed every 48 hours (total of 2 additional media changes). At day 15, cells were dissociated using TrypLE and split to a density of 0.4 x 106 cells/cm2 onto Matrigel coated dishes. Cells were replated in RPMI media (no glucose, Cellgro#10-043-CV) supplemented with 5% FBS, 10 mM D-galactose (Sigma-Aldrich# G5388), 1 mM Sodium Pyruvate (LifeTechnologies# 11360070), 1X NEAA (LifeTechnologies# 11140076), 5 mM HEPES (LifeTechnologies# 15630080), mM Sodium D/L Lactate (Sigma-Aldrich# L4263), 0.5 mg/mL BSA (Sigma-Aldrich# A2153), 0.213 mg/mL ascorbic acid (Santa Cruz Bio# sc-228390) and 1x Pen/Strep (Galactose media). After 24 hours, the media was changed to Galactose media without FBS. Media was changed every 48 hours (total of 1 additional media change). At day 20 media was changed to Galactose media supplemented with 3 ng/mL Triiodothyronine (T3, Sigma-Aldrich# T6397). Media was changed every 48 hours until cells were harvested at day 27 (total of 3 additional media changes). For the first two media changes (Days 0 and 2) cultures were supplies with an excess of media (0.499 mL/cm2), all other days media was added at (0.2495 mL/cm2).

Growth protocol

Feeder free iPSC cultures were initially maintained on Growth Factor Reduced Matrigel using Essential 8 Medium (E8) as previously described. After 10 passages in E8, all cell lines were transitioned to a 50/50% ratio of iDEAL/E8 feeder free medium that was prepared in house as specified previously (Marinho et al. 2015). Cell culture was conducted at 37°C, 5% CO2, and atmospheric O2.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using the ZR-Duet DNA/RNA MiniPrep kit (Zymo) with the addition of an on column DNAse I treatment step prior to RNA elution.
Samples were pooled together in 8 pools for RNA-seq library generation using the Illumina TruSeq RNA Library Prep kit v2. 100 bp sing-end RNA-seq libraries were sequenced on the HiSeq4000 following the manufacturer's protocols.Samples were pooled in 8 pools for RNA-seq library generation using the Illumina TruSeq RNA Library Prep kit v2. 100 bp sing-end RNA-seq libraries were sequenced on the HiSeq4000 following the manufacturer's protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

100 bp single-end RNA-seq reads from each species were mapped to the appropriate genome (hg38 or panTro5) using Hisat2 (version 2.0.4) (Kim et al. 2015). Only reads that mapped uniquely were used.
The number of reads falling into orthologous meta-exons across 44,125 Ensembl genes from hg38, panTro5 was determined using featureCounts within SubRead (version 1.5.1) (Liao et al. 2014).
Genome_build: hg38 & panTro5
Supplementary_files_format_and_content: RNA-seq counts for orthologous genes for all individuals

Submission date

Feb 12, 2018

Last update date

Aug 08, 2018

Contact name

Bryan J Pavlovic

Organization name

University of Chicago

Department

Human Genetics

Street address

920 E 58th St.