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Sample GSM6340920

Query DataSets for GSM6340920

Status

Public on Jan 30, 2023

Title

TABLE-seq_RNA3ng_rep2

Sample type

SRA

Source name

Embryonic stem cells

Organism

Mus musculus

Characteristics

cell type: E14
genotype: WT

Growth protocol

E14 mouse embryonic stem cells (mESCs) were cultured on 0.1% gelatin-coated dishes in DMEM supplemented with 15% fetal bovine serum (FBS), 1% antibiotic solution (penicillin/streptomycin), 1% glutamax, 1% MEM nonessential amino acids, 1% sodium pyruvate, 0.1 mM beta-mercaptoethanol and 1000 U/ml recombinant leukemia inhibitory factor (LIF). HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic solution (penicillin/streptomycin). All cells were grown at 37 C and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

RNA extraction was performed by RNeasy Plus Mini Kit (Qiagen, Cat. #74134).
2 μl 100 μM 5ph_Tn5a and 2 μl 100 μM Tn5a_N6_invert_dT were added to 16 μl H2O. Samples were annealed by 95 C for 5 min and ramped to 25 C at 5 C/min. 1 μl Exo I, 3 μl 10X Exo I reaction buffer, and 6 μl H2O were added and incubated at 37 C for 30 min, followed by 80 C for 15 min. The annealed adaptors were purified by 2X of VAHTS DNA clean beads, eluted into 20 μl H2O, and stored at -20 C until usage. 10 μl tagmented ssDNA or cDNA was mixed with 2 μl annealed adaptors, 0.5 μl T4 DNA ligase (Takara, Cat. #2011A), 2 μl 10X ligation buffer, 2.5 μl 40% PEG6000, 3 μl H2O, and incubated at 20 C for 1 hour. The ligated DNA was purified by 0.8X VAHTS DNA clean beads and eluted into 10 μl H2O. The purified DNA was amplified by primers with sequencing indexes. 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer, 8 μl DNA, and 10 μl 2X HIFI PCR Master Mix (NEB, Cat. #M0541L) were mixed and amplified at 72 C for 5 min, 98 C for 30 sec, 20 cycles of 98 C for 10 sec and 63 C for 10 sec, then 72 C for 1 min, 16 C to hold. PCR products were purified by 0.9X VAHTS DNA clean beads and eluted into 20 μl H2O. The concentrations of DNA were detected by Equalbit 1X dsDNA HS Working Solution (Vazyme, Cat. #121-01-AA). The libraries were sequenced on an Illumina NovaSeq platform with pair-end reads of 150 bp.
TABLE-seq

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

Sequencing reads adaptor and low-quality bases were removed by Trim Galore (version 0.6.4) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore)
Bowtie2 (version 2.3.5.1) was used to map trimmed reads to the mouse reference genome mm9 and Tsin-H3.1-flag plasmid construct reference. PCR duplicates were removed by GATK4 (version 4.1.4.0) (https://github.com/broadinstitute/gatk) with the parameter ‘--REMOVE_DUPLICATES = true’ in cDNA data.
Expression matrixes were counted by featureCounts(Liao et al., 2014) (version 2.0.0)
BEDtools (version 2.92.2) and bedGraphToBigWig (version 4) (https://www.encodeproject.org/software/bedgraphtobigwig/) were used with the following parameters ‘genomecov --scaleFactor 10,000,00/(the number of reads mapped to mm9 or Tsin-H3.1-flag plasmid construct reference)’. Normalized signals were visualized in Integrative Genomics Viewer (IGV) (version 2.6.3).
Assembly: mm9
Assembly: Tsin-H3.1-flag plasmid construct reference
Supplementary files format and content: BigWig files
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample of TABLE-seq

Submission date

Jul 15, 2022

Last update date

Jan 30, 2023

Contact name

Dong Fang

E-mail(s)

dfang@zju.edu.cn

Organization name

Zhejiang University

Department

Life Sciences Institute