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Sample GSM4419014

Query DataSets for GSM4419014

Status

Public on May 26, 2020

Title

Untransfected, Bulk RNA-seq, Replicate 2

Sample type

SRA

Source name

Mouse embryonic stem cells

Organism

Mus musculus

Characteristics

strain: 129P2/OlaHsd
cell type: Mouse embryonic stem cells

Treatment protocol

To induce differentiation, cells were grown for 48 hours in mESC media without GSK3 inhibitor XV and UO126 and then seeded at 104 cells/cm2 in mESC media without GSK3 inhibitor XV and UO126. After 24 hours, media was replaced with serum-free differentiation media: Advanced DMEM supplemented with N-2, B27 Supplement without vitamin A, and Glutamax (Thermo Fisher). After 24 hours, media was replaced with differentiation media: Advanced DMEM with 2% ES-tested FBS and Glutamax. After 24 hours in differentiation media, cells were treated in differentiation media with combinatorial treatment conditions for 24 hours. To induce mesendoderm differentiation, cells were treated for 24 hours in differentiation media with CD 2665, Activin A, GSK3 inhibitor XV, Dorsomorphin, and Fgf2 and then exposed to combinatorial treatment conditions for 24 hours in differentiation media. Control conditions did not receive combinatorial treatments.
Transfection of barcodelets was performed using TransIT-mRNA (Mirus) 2-16 hours before harvest for single cell RNA-seq. Each well of a 96-well plate received 200 ng total barcodelet RNA mixed equally among the 2-5 component barcodelet species. Barcodelets for each well were diluted in 5 µL OptiMEM with 0.1 µL mRNA-Boost reagent. This mixture was mixed well with a mixture of 5 µL OptiMEM + 0.1 µL TransIT-mRNA reagent, incubated at room temperature for 2-5 minutes, and pipetted onto cells in differentiation media with combinatorial treatment conditions.

Growth protocol

Culture and differentiation of 129P2/OlaHsd male mouse embryonic stem cells was modified slightly from previously published protocols (Sherwood et al., 2014). mESCs were grown at 37 degrees Celsius in mESC media: Knockout DMEM supplemented with 15% ES-tested fetal bovine serum, 0.1 mM nonessential amino acids, Glutamax, 0.55 mM 2-mercaptoethanol (all from Thermo Fisher), 1X ESGRO LIF, 5 µM GSK3 inhibitor XV, and 50 µM UO126 (Millipore).

Extracted molecule

polyA RNA

Extraction protocol

For Foxa2GFPplus, Foxa2GFPminus and Tdgf1GFPplus samples, cell populations were flow cytometrically isolated prior to RNA isolation
3’-enriched RNA-sequencing was performed using the Lexogen Quantseq 3’ mRNA-seq kit using manufacturer-suggested protocols and sequenced using Illumina Nextseq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

barRNA-seq transcriptome reads were mapped using the 10X cellranger pipeline (v1.3.1) to mm10 (v.1.2.0).
barRNA-seq barcodelet reads were mapped using the 10X cellranger pipeline (v1.3.1) modified with a custom mapping step that first aligns and trims the common right flanking sequence, and then maps the remaining sequence to the known pool of unique barcodelet sequences. The alignment allows for 4 mismatches in the right flanking sequence, and 2 mismatches in the unique subsequence. The output of the pipeline is UMI-unique barcodelet counts for each cell.
To assign each cell a treatment combination, we inspect the top k most abundant barcodelet species in the count data, where k is the number of barcodelets each cell was labeled with, and compute the summed count fraction of these top k barcodelets. We expect that invalid combinations observed are due to noise. Since we also know the expected fraction of invalid combination based on the size of the starting pool of barcodelets, we can estimate the false positive rate at any given summed count fraction. To ensure labeling fidelity, we set the threshold on the summed count fraction at a false positive rate of 1%, and assign all cells above that threshold for which the barcodelet combination was valid. We retain the set of cells which have been assigned a treatment combination and that also have a high quality transcriptome for downstream analysis.
Bulk RNA-seq reads were mapped using the Quantseq 3' mRNA mapping pipeline as described by lexogen to mm10. Briefly, reads were first trimmed using bbduk from the bbmap suite (v37.75) trimming for low quality tails, poly-A read-through and adapter contamination using the recommended parameters. Then, reads were mapped using the STAR aligner (v2.5.2b) with the recommended modified-Encode settings. Finally, HT-seq (v0.9.1) count was used to obtain per-gene counts.
Genome_build: mm10
Genome_build: germlayer_bcd.fa (reference for germlayer_bcd.possorted_genome_bam.bam)
Genome_build: mesendoderm_bcd.fa (reference for mesendoderm_bcd.possorted_genome_bam.bam)
Genome_build: mixed_bcd.fa (reference for mixed_bcd.possorted_genome_bam.bam)
Supplementary_files_format_and_content: Gene-barcode matrices are stored as matrices in *mtx files, with gene and barcode metadata in *tsv files. These are generated by 10X, and more information on the format can be found here: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/1.3/output/matrices. Filtered gene-barcode matrices are provided for transcriptomic reads, while raw gene-barcode matrices are provided for the barcodelet reads.
Supplementary_files_format_and_content: Treatment group assignments are stored in *assignments.csv files.

Submission date

Mar 17, 2020

Last update date

May 26, 2020

Contact name

Richard I Sherwood

Organization name

Brighan and Women's Hospital and Harvard Medical School

Department

Division of Genetics, Department of Medicine

Street address

77 Avenue Louis Pasteur