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Sample GSM1486874

Query DataSets for GSM1486874

Status

Public on Dec 04, 2014

Title

TwoiLIF_A6

Sample type

SRA

Source name

v6.5 mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell type: v6.5 mouse embryonic stem cells
culture conditions: 2i+LIF

Growth protocol

Described under 'culture conditions' in sample information.

Extracted molecule

total RNA

Extraction protocol

mES or neural precursor cells were taken directly from culture, trypsinized, washed in PBS, spun down and resuspended at a concentration of 3 x 10^5 cell per mL of complete media, mixed 7:3 with C1 suspension reagent (Fluidigm), and loaded onto C1 Single-Cell Auto Prep chips (C1 chips; Fluidigm). After loading, each of the cell isolation chambers on the C1 chip was optically inspected for the presence of a cell. Subsequently, these cells were lysed and SMART-Seq whole transcriptome amplified (WTA) products were prepared with the C1 System using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) and protocols provided by Fluidigm (full details available at www.fluidigm.com).
WTA products were harvested from the C1 chip, diluted to a concentration of 0.15 ng/μL, and cDNA libraries were prepared using Nextera XT DNA Sample preparation reagents (Illumina) as per the manufacturer’s recommendations, with minor modifications. Specifically, reactions were run at ¼ the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30s to 60s. After the PCR step, all 96 samples were pooled without library normalization, cleaned twice with 0.9x AMPure XP SPRI beads (Beckman Coulter), and eluted in buffer TE. The pooled libraries were quantified using Quant-IT DNA High-Sensitivity Assay Kit (Invitrogen) and examined using a high-sensitivity DNA chip (Agilent). Finally, samples were sequenced deeply using either a HiSeq 2000 or a HiSeq 2500 sequencer (25 bp paired-end reads).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

TwoiLIF_A6
RnaSeq_single_cell_mESC_TPM.csv

Data processing

Alignment: A Bowtie index was created based on the UCSC knownGene transcriptome, and paired-end reads were directly aligned to this index.
Calculation of expression levels: RSEM was run on these alignments to generate expression level estimates, and these estimates (tau) were multiplied by 1,000,000 to yield estimates of transcripts per million (TPM) for each gene. These TPM estimates were then transformed into log space by taking ln(TPM+1), which we hereafter refer to in the Supplement as ln(TPM).
Genome_build: mm9
Supplementary_files_format_and_content: For single-cell RNA-Seq, tab-delimited text files providing RSEM ln-transformed transcript per million (TPM) values for each sample. For ChIP-Seq, total number of reads mapping to promoter regions +1500 bp to -500 bp from annotated transcription start sites.

Submission date

Aug 26, 2014

Last update date

May 15, 2019

Contact name

Patrick Cahan

E-mail(s)

patrick.cahan@jhmi.edu

Organization name

Johns Hopkins University School of Medicine

Department

ICE and Biomedical Engineering