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| Status |
Public on Oct 19, 2021 |
| Title |
RNA_EB_Day6_CaPS_R_Rep2 |
| Sample type |
SRA |
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| Source name |
Embryoid bodies
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| Organism |
Mus musculus |
| Characteristics |
cell type: J1 cells
genotype: CBX7-CaPS-R
age: 6 days
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| Growth protocol |
Embryonic stem cells (ESCs) were cultured on 1% gelatin with a feeder layer of MEFs in DMEM supplemented with 15% Hyclone FBS (GE Healthcare), GlutaMax (Invitrogen), penicillin/streptomycin, non-essential amino acids, 1000U/mL LIF, and 2-betamercatoethanol. To form embryoid bodies (EBs), ESCs were de-MEFed and resuspended in differentiation media (IMDM supplemented with 15% HyClone FBS , GlutaMax, and penicillin/streptomycin) at a concentration of 8,400 cells/mL. Drops were set at 50uL per drop on the bottom of a non-adherent tissue culture plate, and plates were storeed upside down. After 4 days, EBs were pooled together and grown in suspension in non-adherent plates, changing media every day. EBs were collected for analysis on days 6 and 10 of differentiation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from pelleted cells using the PureLink RNA Mini Kit (Invitrogen) according to manufacturer's instructions. Samples were treated with DNase using the TURBO DNA-free Kit (Invitrogen). rRNA was depleted using the NEBNext rRNA Depletion Kit, and cDNA was generated using the NEBNext Ultra II Directional RNA First and Second Strand Synthesis Modules (New England Biolabs).
Libraries were prepared as described in Bowman et al., 2013 (BMC Genomics). In brief, this protocol includes end repair, A-tailing, adapter ligation, and real-time PCR amplification, with SPRI cleanup of samples between each step.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
CaPS_R_EB_counts.txt
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| Data processing |
Adapter sequences were removed from reads usinng Cutadapt v.1.14 with "--minimumLength 30"
Reads were aligned to the mm10 genome using STAR v2.7.0
Reads in gene exons were counted using featureCounts v1.6.3
Gene annotations were obtained from ncbi refseq
EdgeR was used for differential gene expression analyses
Genome_build: mm10
Supplementary_files_format_and_content: Genome browser tracks were generated using Homer v4.10.4
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| Submission date |
Oct 18, 2021 |
| Last update date |
Oct 19, 2021 |
| Contact name |
Robert Kingston |
| E-mail(s) |
kingston@molbio.mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital
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| Department |
Molecular Biology
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| Lab |
Kingston Lab
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| Street address |
185 Cambridge Street, 7th Floor
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE151900 |
A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells (EBs/NPCs RNA-seq datasets) |
| GSE151901 |
A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells |
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| Relations |
| BioSample |
SAMN22374035 |
| SRA |
SRX12677614 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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