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Sample GSM4323252

Query DataSets for GSM4323252

Status

Public on Dec 22, 2020

Title

278_2i_Mthfd1l

Sample type

SRA

Source name

embryonic stem cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype: Mthfd1l KO
medium: 2i
time point: 0 h

Treatment protocol

Differentiation medium (N2B27) consisted of a 1:1 mixture of DMEM/F12 and Neurobasal medium supplemented with 1x B27 (Gibco), 0.5x N2 (homemade), 0.1mM NEAA, 1mM L-glutamine, 1x Penicillin-Streptomycin and 0.05mM 2-mercaptoethanol. 24h before starting differentiation, medium was changed from maintenance ESDMEM-2i medium to N2B27 medium supplemented with 3µM CHIR99201 and 1µM PD0325901 (N2B27-2i). Cells were differentiated in parallel in duplicates for 2-32 hours in unsupplemented N2B27. The undifferentiated control samples were cultured in N2B27-2i.

Growth protocol

Cells were maintained in DMEM supplemented with 15% FCS (batch tested, Biowest), 1x Penicillin-Streptomycin (Sigma), 0.1mM NEEA, 1mM sodium pyruvate, 1mM L-glutamine, 0.05mM 2-mercatpoethanol (Gibco), 10ng/mL LIF (batch tested, in-house), 1.5µM CHIRON and 0.5µM PD0325901 (ESDMEM-2i) on gelatin coated plates.

Extracted molecule

total RNA

Extraction protocol

RNA was extraced with the RNeasy Mini Kit (Qiagen) according to the manufacturer`s protocol.
[1] Single end data: mRNA was isolated from 1 ug total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding beads in a ratio of 1:1, followed by an index PCR (15 cycles) using Illumina compatible index primer. After double XP beads purifications (with beads added in a ratio 1:1) libraries were quantified on a Fragment Analyzer run with a NGS Assay Kit (Agilent) and loaded on a HiSeq 3000 flowcell with 50 cycles single end seqeuncing by pooling the samples in respect of their molarity aiming at 30 mio. reads per sample.
[2] paired-end data: Illumina TruSeq mRNA stranded

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

[1]
counts_KOs.csv
FPKM_KOs.csv
L11981

Data processing

STAR (version 2.5.3) was used to align single end reads and to retrieve raw counts
STAR(version 2.5.2b) was used to align paired end reads and to retrieve raw counts
FPKM values were calculated using DeSEQ2 (version 1.20.0).
TPM values were calculated using exon sizes from gencode.vM11.primary_assembly.annotation.gtf
Genome_build: mm10
Supplementary_files_format_and_content: CSV (raw counts of single and paired end data in separate files; FPKM of single end data; TPM of paired end data)

Submission date

Feb 20, 2020

Last update date

Dec 22, 2020

Contact name

Andreas Beyer

E-mail(s)

[email protected], [email protected]

Organization name

University of Cologne

Department

CECAD