|
Status |
Public on Dec 20, 2012 |
Title |
Day3_SSEA1+ M2 K4 |
Sample type |
SRA |
|
|
Source name |
Sorted cells
|
Organism |
Mus musculus |
Characteristics |
mark: K4 chip antibody: Anti-Trimethyl Histone H3 (Lys4) Antibody, (Millipore, clone: CMA304, catalog#: 05-1339, lot#: 2024897) time after induction[days]: 3 cell stage: Intermediates thy1: NEG ssea1: POS gfp: NEG
|
Treatment protocol |
Harvested cells were incubated with antibodies against Thy1.2 (PE conjugated, 53-2.1, eBiosciences) and SSEA-1 (mouse IgM, MC-480, Developmental Hybridoma Bank) for 20 minutes. Cells were washed in PBS and then incubated for 20 minutes with APC conjugated anti mouse IgM, (eBioscience) and Pacific Blue-conjugated streptavidin (Invitrogen). The cells were washed in PBS, resuspended in propidium iodide 5% FBS/PBS solution and passed through a 40mm cell strainer to achieve single cell suspension. Cells were sorted on a FACSAria (BD Biosciences ) and/or analysed in a LSRII (BD Bioscience). For analysis and/or sorting of intermediates, cells were stained with Thy1.2 and SSEA1 antibodies and sorted or analyzed as indicated.
|
Growth protocol |
Mouse Embryonic fibroblast (MEFF) cultures were established from E13.5 embryos from a reprogrammable mice strain carrying one copy of the OKSM cassette and Rosa26-M2rtTA allele (het/het) or carrying two copies of the OKSM cassette and Rosa26-M2rtTA allele (ho/ho). Reprogramming was performed in ESC medium (KODMEM with 15% FBS, L-Glutamin, penicillin-streptomycin, non-essential amino acids, b-mercaptoethanol and 1000 U/ml LIF) in the presence of doxycycline.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
iPSCs were harvested when they reached about 50% confluency and preplated on non-gelatinized T25 flasks for 45 minutes to remove feeder cells. Intermidiates cell types were harvest after FACS sorting. ChIP complexes were eluted with 1% SDS in 100 mM NaHCO3 and crosslinks were reversed by incubating samples over night at 65C. DNA fragments were purified using the QIAquick PCR Purification Kit Cat number: 28104. Libraries for sequencing were prepared using ChIP-Seq Sample Prep Kit from Illumina, Cat IP-102-1001
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|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
Sample name: KH181.K4 ChIPed DNA *p.cov.bigWig: processed data file (plus strand). *m.cov.bigWig: processed data file (minus strand).
|
Data processing |
alignment using bwa coverage using genomeCoverageBed in bedtools conversion of coverage to bigWig format using bedGraphToBigWig Genome_build: mm9 Supplementary_files_format_and_content: content is coverage (i.e., number of reads aligning at each position in the genome) and format is bigWig (see genome.ucsc.edu/goldenPath/help/bigWig.html)
|
|
|
Submission date |
Nov 23, 2012 |
Last update date |
May 15, 2019 |
Contact name |
Ben S. Wittner |
E-mail(s) |
wittner.ben@mgh.harvard.edu
|
Organization name |
Massachusetts General Hospital
|
Department |
Center for Cancer Research
|
Lab |
Lawrence
|
Street address |
149 13th Street
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02129 |
Country |
USA |
|
|
Platform ID |
GPL13112 |
Series (2) |
GSE42477 |
Defining a molecular roadmap of cellular reprogramming into iPS cells [ChIP-Seq] |
GSE42478 |
Defining a molecular roadmap of cellular reprogramming into iPS cells |
|
Relations |
SRA |
SRX206138 |
BioSample |
SAMN01818503 |
Named Annotation |
GSM1040719_EAa69.m.cov.bigWig |
Named Annotation |
GSM1040719_EAa69.p.cov.bigWig |