|
| Status |
Public on Oct 05, 2021 |
| Title |
BXD45-RNA-ESC |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cell
|
| Organism |
Mus musculus |
| Characteristics |
cell type: naive esc
mouse strain: bxd
|
| Growth protocol |
Mouse embryonic stem cells were derived and maintained in naive conditions following protocol from Czechanski et al. 2014 (Nature Protocols). Culture conditions to transition ESCs to EpiLCs followed Buecker et al. 2014 (Cell Stem Cell). Spontaneous differentiation of ESCs to EBs was achieved by first transitioning through EpiLC state for 48 hours followed by seeding cells on AggreWell400 Plates (STEMCELL Technologies) in EB medium ensuring 750 cells/EB. Aggregates were allowed to form for 48 hours and then filtered to remove unincorporated cells. Collected EBs were then plated on 100mm Corning Ultra-low attachment culture dishes and continued growth with rotation on BellyButton rotator for another 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A single cell suspension of ESCs and EpiLCs were harvested at a concentration of 1 million cells/mL; 1 million cells were lysed and RNA extracted using RNeasy (Qiagen) RNA extraction kit; 100,000 cells were used for transposition reaction. For ChIP, 10 million cells were harvested and nuclei isolated using hypotonic lysis. Chromatin was fragmented using Covaris followed by dialysis and immunoprecipitation against protein targets was performed. For single cell RNAseq, single cell suspension was achieved for EB cultures and 500,000 cells for each individual sample was labeled with unique Multiseq LMO barcode. Labelled samples were pooled at 3,000 cells per sample and 40,000 cells were loaded onto a single lane of a 10X Chromium microfluidic chip. Cells were lysed in droplets using 10X Chromium instrument and reverse transcription was performed in droplet and library construction performed according to 10X Chromium v3 chemistry.
Libraries were constructed using the KAPA mRNA HyperPrep Kit for ESC and EpiLC bulk RNA samples. Modified Illumina Nextera adapters were used to construct ATAC libraries. ChIP-seq librariers were constructed using KAPA HyperPrep Kit. Single cell RNA-seq library preparation was performed using 10X Chromium v3.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
esc_counts.norm.tsv.gz
|
| Data processing |
Bulk RNA reads were aligned to transcriptome (mm10 or modified to include known D2 SNPs, R78-REL1505) using bowtie v.1.0.0. ChIP and ATAC reads were aligned to genome using hisat2 v.2.0.5.
Allele specific gene counts were performed using EMASE. For ChIP and ATAC, peaks were called using MACS v.1.4.2.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited txt files representing ensembl gene id along with normalized read counts for each sample. tab-delimited txt files representing the position of identified ChIP peaks in mm10 coordinates along with normalized read counts. Expression libraries for single cell RNAseq gene-cell: matrices are provided in the format produced by CellRanger. MULTI-seq Libraries for single cell RNAseq: barcode-cell matrices are provided in CSV format. The columns in the matrix Byers_EB_lipidhash_deMULTIplex_matrix.csv.gz represent individual embryoid body samples identified by unique lipid-modified oligos.
|
| |
|
| Submission date |
Jan 15, 2021 |
| Last update date |
Oct 05, 2021 |
| Contact name |
Christopher Baker |
| E-mail(s) |
christopher.baker@jax.org
|
| Organization name |
The Jackson Laboratory
|
| Street address |
600 Main St
|
| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE164935 |
Genetic control of pluripotency epigenome informs differentiation bias in mouse embryonic stem cells |
|
| Relations |
| BioSample |
SAMN17345722 |
| SRA |
SRX9858125 |
