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Sample GSM6754198

Query DataSets for GSM6754198

Status

Public on Jan 30, 2023

Title

ccTSC.01_p10_Rep_2

Sample type

SRA

Source name

TSC derived from KiPS primed-iPSCs

Organism

Homo sapiens

Characteristics

cell type: human embryonic stem cells
growth protocol: hTSCs were cultured in 12-well plate coated with 5 μg/mL Collagen IV (Corning, 354233) at 37°C overnight or on mitotically inactivated mouse embryonic fibroblasts. Cells were cultured in 0.8 mL TS medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.2% FBS, 0.5% Penicillin-Streptomycin, 0.3%, BSA (Gibco 15260-037), 1% ITS-X (Gibco, 51500), 1.5 μg/ml L-ascorbic acid (Sigma A4544), 50 ng/ml EGF (peprotech AF-100-15), 2 μM CHIR99021 (Axon Medchem cat. nos 1386 and 1408), 0.5 μM A83-01 (Axon Medchem 1421), 1 μM SB431542 (Axon Medchem 1661), 0.8 mM VPA (HDACi, Sigma, P4543), and 5 μM Y-27632] and in 5% CO2 and 5% O2. Media were changed every 2 days, and cells were passed using TrypLE Express every 3-4 days at a ratio of 1:8.
treatment protocol: Chemical resetting from primed to naive PSC. Cells were seeded at 10000 cells/cm2 on mitotically inactivated MEFs in E8 medium with 10µM ROCKi (added only for 24 hours). Two days after plating (day 0), medium was changed to PDL/HDACi (N2B27 with 1µM PD0325901, 10ng/ml human LIF and 1mM VPA (HDACi)). Following 3 days in PDL/HDACi, medium was changed to PXGL for a further 10-11 days before passing in PXGL medium on MEFs (see Fig. 1a). For the chemical resetting from primed to ccTSCs, KiPS were seeded at 10000 cells/cm2 on mitotically inactivated MEFs in E8 medium. Two days later (day 0) medium was changed to PDL/HDACi. Following 3 days in PDL/HDACi, medium was changed to PXGL.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using Total RNA Purification kit (Norgen Biotek), according to manufactorer's protocol.
Quant Seq 3' mRNA-seq Library Prep kit (Lexogen) is used for library construction.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

raw_counts_matrix_2.txt

Data processing

trimming was performed using BBDuk from BBTools v38.87
alignment was performed with STAR 2.7.6a
quantification was performed with Salmon v1.6.0
Assembly: Human GRCh38.p13 Ensembl release 105
Supplementary files format and content: raw_counts_matrix_2.txt is a txt containing EnsemblID, official gene symbols and raw counts for each condition

Submission date

Nov 23, 2022

Last update date

Jan 30, 2023

Contact name

Graziano Martello

E-mail(s)

graziano.martello@unipd.it

Organization name

University of Padua

Department

Department of Molecular Medicine