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Sample GSM4891025

Query DataSets for GSM4891025

Status

Public on Dec 01, 2021

Title

Rhino-lib13: NWR iPSCs primed - rep2

Sample type

SRA

Source name

iPSCs

Organism

Ceratotherium simum cottoni

Characteristics

cell line: NWR iPSCs
culturing conditions: primed
medium: mTeSR1
feeder: non

Growth protocol

Primed culturing conditions: NWR iPSCs were maintained feeder-free on Matrigel coated 6-Well plates in chemically defined mTeSR1 medium (STEMCELL technologies, #85850) supplemented with ascorbic acid (50 µg/ml, Sigma-Aldrich, #A8960-5G) at 37°C, 5% CO2 and 5% O2. Cells were split every 3 to 4 days (confluency ~80 %) using PBS 0.5 mM EDTA solution (Life technologies, #14190-250 and Thermo Fisher, #15575-020) in ratios of 1:6 and 1:12. Naive culturing conditions: To induce the naive state, cells were transitioned from primed conditions either to RSet (Stemcell Technologies, #05970) or N2B27 medium [1:1 DMEM/F12:Neurobasal medium (#11320-033 and #21103-049), 1x N2-Supplement (#17502-048), 1x B27-Supplement w/o Vit. A (#12587-010), 1x GlutaMAX (#35050-038), 1x MEM NEAA (#11140-035), 0.11 mM ß-Mercaptoethanol (#21985-023), 10 ng/ml LIF (Merck/Millipore, #LIF1010), 0.075 µM Bio (Tocris Bioscience, #3194), 5 µM XAV (Tocris Bioscience, #3748), 0.5 µM PD-0325901 (BioVision, #1643), 2 µM Gö6983 (Tocris Bioscience, #6983), 75 µg/ml ascorbic acid (Sigma-Aldrich, #A8960), 15ng/ml Activin A (#PHC9564). If not indicated otherwise, all medium components were from Life Technologies.] as follow: After TrypLE split (TrypLE 1x, Life Technologies, #12563-011, ratio 1:6 and 1:12), primed iPSCs were plated as single cells on 500.000 MEFs/6-Well (inactivated mouse embryonic fibroblasts, teBu-bio, #003MEF-MITC) with 10 µM ROCKi (ROCK Inhibitor Y-27632 2HCl; 10mM/1mL, Selleck Chemicals, #SEL-S1049-10MM) and incubated overnight in mTeSR1 (NWR iPSCs) or E8 medium (human BIHi005A). At the next day, medium was changed to N2B27 or RSet. To facilitate the conversion process, the NWR iBCL2-GFP-iPSC line was generated (see above) and ectopic expression of BCL2 was induced by 1 µg/ml Doxycycline (Clontech, #631311). Cells were maintained on MEFs in N2B27 or rather RSet medium at 37°C, 5% CO2 and 5% O2, and split using TrypLE every 3 to 4 days (confluency ~80%) in ratios varying from 1:4 to 1:10.

Extracted molecule

total RNA

Extraction protocol

Total RNA was was isolated using RNeasy mini kit (Qiagen, #74104) according to the manufacturer’s instructions. 3 µg of isolated RNA were treated with TURBO DNase (Life Technologies, #AM2239) followed by clean up using RNeasy MinElute RNA cleanup kit (Qiagen, #74204). RNA purity and quality were assessed with microcapillary electrophoresis on Agilent 2100 Bioanalyzer (RNA Pico 6000 kit, Agilent, #5067-1513). Ribosomal RNA was depleted from 1 µg of total RNA using RiboZero Gold (Human/Mouse/Rat) kit (Illumina), followed by purification with RNeasy MinElute RNA cleanup kit.
Sequencing libraries were prepared from rRNA-depleted RNA using TruSeq Stranded total RNA LT kit (Illumina, #20020598) according to the manufacturer’s instructions. The final PCR amplification included 11 cycles followed by purification with Ampure XP beads (Beckman-Coulter, #A63881). Quality of final libraries was evaluated by Agilent 2100 Bioanalyzer using DNA 1000 kits (Agilent, #5067-1504). DNA concentrations were measured with Qubit (Life Technologies) and dsDNA HS Assay Kit (Thermo Fisher Scientific, #Q32854). Libraries were sequenced using Illumina NextSeq 500 instrument [75-nt single-end reads, NextSeq 500/550 High Output Kit v2.5 (75 Cycles), Illumina, #20024906].

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

RNA-seq sequencing reads were clipped for residual adapter sequences and trimmed for low-quality 3’ ends.
For mapping, we used STAR (v.2.7.1a) (Dobin et al. 2013) to align all RNA-seq datasets to the SWR genome. Maximum number of mismatches was set up to 4 to account for subspecies variability. Multiple mapping to several locations was allowed unless otherwise stated.
We quantified the gene expression by using HTSeq (Anders et al. 2015) and we filtered out genes with an average FPKM lower than 1. Read counts were normalized by sample by applying DESeq2’s median of ratios (Love et al. 2014).
Genome_build: cerSim1
Supplementary_files_format_and_content: tsv file, include raw counts per sample and gene
Supplementary_files_format_and_content: gtf file, include the transcriptome used in this study

Submission date

Nov 10, 2020

Last update date

Dec 01, 2021

Contact name

Jorge Ruiz-Orera

E-mail(s)

jorruior@gmail.com

Organization name

Max Delbruck Center for Molecular Medicine

Street address

Robert-Rössle-Straße 10