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| Status |
Public on Oct 12, 2015 |
| Title |
20130423_H3K4me2_12 |
| Sample type |
SRA |
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| Source name |
20130423_H3K4me2
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| Organism |
Mus musculus |
| Characteristics |
cells pointed by barcodes: mESC: 1-576; MEF: 577-1152
mescs strain: V6.5 strain
mefs source: Globalstem:GSC-6002
eml source: NA
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| Growth protocol |
mESCs were grown using Knockout DMEM: ~410mL (Gibco) 15% Fetal Bovine serum: 75mL (lot# F12101121) 1% Pen/Strep: 5mL (Gibco) 1% Non-essential amino acids: 5mL (Gibco) 1% Glutamax: 5mL (Gibco) .01% LIF: 50ul (Millipore: ESG1107) .0004% BME: 2ul mEFs were grown using Knockout DMEM: ~410mL (Gibco) 15% Fetal Bovine serum: 75mL (lot# F12101121) 1% Pen/Strep: 5mL (Gibco) 1% Non-essential amino acids: 5mL (Gibco) 1% Glutamax: 5mL (Gibco)
The mEFs were grown:using Knockout DMEM: ~410mL (Gibco) 15% Fetal Bovine serum: 75mL (lot# F12101121) 1% Pen/Strep: 5mL (Gibco) 1% Non-essential amino acids: 5mL (Gibco) 1% Glutamax: 5mL (Gibco)
EML were grown as in Hannah H. Chang et. al."Transcriptome-wide noise controls lineage choice in mammalian progenitor cells"
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were trypsinized and washed prior to encapsulation
To minimize the abundance of barcode adaptors concatemers we use PacI restriction enzyme (R0547L, NEB, USA) immediately after ChIP washing steps and while the chromatin is still bound to the ChIP beads. PacI digest in between bound concatemers and in the middle of each adapter to form 30bp DNA fragments that can be easily filter out using simple size selection. Next, we elute the chromatin by adding 2X elution buffer, digesting RNA contaminates with Rnase (11119915001, Roche Diagnostics, USA) and removing the nucleosomes with ProtenaseK (P8102S, NEB, USA). To purify the DNA we use AMPure XP beads (A63880, Beckman Coulter, USA) and follow with 14 rounds of Single-Cell-PCR to amplify the labeled DNA. To reduce unspecific Illumina adapter ligation we first dephosphorylate all 5’ ends with Antarctic Phosphatase (M0289L, NEB, USA) and then use BciVi enzyme (R0596L, NEB, USA) to specifically cleave the labeled DNA, leaving an A overhang at the 5’ end of all DNA fragments with single cell adapters. After ligating Illumina adapters, we digest concatemers again using PacI and perform 14 additional rounds of Illumina-PCR (PfuUltra II Hotstart PCR Master Mix, 600850, Agilent Technologies, USA) before sequencing the library of reads.
Size selected for 200bp-1000bp
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
36_TTGAATAG
antibody: Millipore 07-030
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| Data processing |
Extracting single cell by barcodes. "Forward_Adapters.xlsx" includes a list of all 1152 barcoded adapters
alignment with Bowtie
filtering duplicates
find mESC clusters using ChIP seq signatures
aggregating mESC clusters
Peak calling
Genome_build: mm9
Supplementary_files_format_and_content: bed
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| Submission date |
Jun 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
oren ram |
| E-mail(s) |
oren@broadinstitute.org
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| Phone |
6178343661
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| Organization name |
Broad institute
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| Department |
epigenomics
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| Lab |
Brad Bernstein
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| Street address |
415 Main Street
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| City |
cambridge |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE70253 |
Single-cell chromatin profiles reveal a spectrum of stem cell epigenetic states |
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| Relations |
| BioSample |
SAMN03793266 |
| SRA |
SRX1073852 |
| 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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