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Sample GSM6754225

Query DataSets for GSM6754225

Status

Public on Jan 30, 2023

Title

KiPS_RESET_D6_TSCmedium_Rep_2

Sample type

SRA

Source name

KiPS-chemical resetting

Organism

Homo sapiens

Characteristics

cell type: chemical reset induced pluripotent stem cells
growth protocol: Cells were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs; DR4 ATCC) in PXGL medium3 or in RSeT medium (Stem Cell Technologies 05969 ). The PXGL medium was prepared as follows: N2B27 (DMEM/F12 [Gibco 11320-074], and Neurobasal in 1:1 ratio [Gibco 21103-049], with 1:200 N2 Supplement [Gibco 17502-048], and 1:100 B27 Supplement [Gibco 17504-044], 2 mM L-glutamine [Gibco 25030-024], 0.1 mM 2-mercaptoethanol [Sigma-Aldrich M3148]) supplemented with 1µM PD0325901 (Axon Medchem), 2 μM XAV939 (Axon Medchem), 2 μM Gö6983 (Axon Medchem) and 10 ng/ml human LIF (produced in-house). Human naive PSCs were passaged as single cells every 4 days at split ratio 1:3 or 1:4 following dissociation with TrypLE (Gibco 12563-029) for 10 minutes at room temperature (RT). The ROCK inhibitor (Y27632, Axon Medchem 1683) was added in the naïve medium only for 24h after passaging.
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