alias: E-MTAB-9053:p59_p
title: Illumina HiSeq 2000 paired end sequencing; Human iPSC-derived cardiomyocytes of peripartum cardiomyopathy patients reveal aberrant expression of sterol and lipid metabolism pathways
design: Human iPSC-derived cardiomyocytes of peripartum cardiomyopathy patients reveal aberrant expression of sterol and lipid metabolism pathways
library_construction_protocol: Skin biopsies were collected by punch biopsy (4 mm in diameter) from the inside of the upper arm. After manually cutting the biopsies to smaller pieces, fibroblasts were allowed to grow out of the pieces of skin by culturing in fibroblast medium consisting of DMEM supplemented with 15% fetal bovine serum (F7524, Sigma-Aldrich), 10% sodium pyruvate (11360070, Thermo Fisher Scientific), 10% non-essential amino acids (11140076, Thermo Fisher Scientific), 10% GlutaMAX (35050061, Thermo Fisher Scientific) and 10% penicillin/streptomycin (15070063, Thermo Fisher Scientific). Once enough fibroblasts were obtained, reprogramming was performed using an Amaxa Nucleofector and a NHDF nucleofection kit (VPD-1001, Lonza). Episomal vectors pCXLE-hOCT3/4, pCXLE-hSK, and pCXLE-hMLN were obtained from Addgene and were a gift from Shinya Yamanaka (Addgene numbers 27076, 27078, and 27079 respectively). Vectors were mixed in a ratio of 2:3:2 respectively in a nucleofection mix with 3 μg plasmids in total.  Nucleofected fibroblasts were cultured in fibroblast medium until cultures reached 70-80% confluency. Then, medium was switched to Essential 6 medium (A1516401, Thermo Fisher Scientific). When the first hiPSC colonies were observed, 10 ng/ml basic fibroblast growth factor (bFGF; 100-18B, Peprotech) was added to the Essential 6 medium. At day 24 after nucleofection, medium was switched to Essential 8 medium (A1517001, Thermo Fisher Scientific). HiPSC colonies were excised from the fibroblast monolayer between days 26-32 after nucleofection. Newly generated hiPSC colonies were initially cultured in Essential 8 medium on vitronectin-coated surfaces (07180, Stem Cell Technologies). After several passages, the culturing surface was switched to Geltrex (A1413301, Thermo Fisher Scientific). Passaging was done by accutase-mediated (A6964, Sigma Aldrich) enzymatic dissociation.  Differentiation to cardiomyocytes was achieved based on a modified protocol as described previously[14]. Briefly, hiPSCs were dissociated with accutase for 3 minutes and plated as single cells in Essential 8 medium containing 10 μM Y26732 (S1049, Selleck Chemicals), Essential 8 medium (without Y26732) was refreshed daily. Once cultures reached 80% confluency, cells were washed with PBS and differentiation was initiated (day 0) by culturing cells in CDM3 medium (as described by Burridge et al.[15]) supplemented with 5% KnockOut Serum Replacement (KOSR; 10828028, Thermo Fisher Scientific), 6 μM CHIR99021 (13122, Cayman Chemical) and 50 ng/ml Activin A (120-14E, Peprotech). At day 2, cells were washed with PBS and medium was refreshed with CDM3 medium supplemented with 5% KOSR, 2 μM Wnt-C59 (5148, Tocris Bioscience) and 5 ng/ml bone morphogenic protein 4 (BMP4; SRP3016, Sigma Aldrich). From day 4, medium was changed to CDM3 medium and was refreshed every other day as cardiomyocyte maintenance medium. This resulted in cultures with >90% spontaneously contracting cardiomyocytes at day 8-10. To further enrich these cultures, starting from day 12, differentiated cardiomyocytes were cultured in glucose-free RPMI1640-based (11879, Thermo Fisher Scientific) CDM3 medium supplemented with 5 mM sodium DL-lactate (CDM3L; L4263, Sigma-Aldrich) for 4 days[15]. This resulted in >99% pure spontaneously beating cardiomyocytes. Experiments were typically started at day 28. Cyclic equiaxial mechanical stretch was induced according a previously published protocol. Briefly, hiPSC-CM were seeded on Geltrex-coated Bioflex 6-well plates (BF-3001C, Flexcell). Mechanical stretch was induced at 15% and 1 Hz for 48 hours using a FX-4000 (FlexCell). Control hiPSC-CM were cultured on the same plates without applying mechanical stretch.  Total RNA was isolated with TRI reagent according to the manufacturer protocol (T9424, Sigma). 1 μg of RNA was used for 150 bp RNA library sequencing (Illumina TruSeq RNA Library Prep Kit ).
identifiers.namespace: 1 Department of Cardiology, University Medical Center Groningen, University of Groningen,
identifiers.id: E-MTAB-9053:p59_p
attributes.tag: Experimental Factor: disease
attributes.value: peripartum cardiomyopathy
attributes.tag: Experimental Factor: stimulus
attributes.value: mechanical stretch 1Hz 15% 48h
attributes.tag: Experimental Factor: pair
attributes.value: 7
attributes.tag: Experimental Factor: family
attributes.value: family A