|
| Status |
Public on Jun 01, 2013 |
| Title |
IgH_IgMb_D4_C_reg_145501_FVB_Cy3 |
| Sample type |
SRA |
| |
|
| Source name |
Activated, mature B cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB
age: 6-8 wks
tissue: spleen
cell type: in vitro activated, Splenic B cells
sequencing: Paired-end
|
| Treatment protocol |
cells are cross linked using 2% formaldehyde for 10minutes at room temperature in 10%FCS/PBS
|
| Growth protocol |
Resting mature B and T cells are isolated from the spleen from 6-8 weeks mice and immediately processed (Day 0). To obtain activated mature B and T cells, resting splenic B and T cells are isolated and subsequently in vitro activated for 4 days after which they are processed (Day 4). Fetal brain cells are isolated at embryonic stage e14.5.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The initial steps of the 4C procedure, as published before (Simonis et al., 2006, Nature Genetics), remain unchanged. Cells are cross linked using 2% formaldehyde for 10min at room temperature in PBS 10%FCS, nuclei are isolated, after which chromatin is digested with HindIII and subsequently ligated under diluted conditions. After reversal of the cross links the DNA is purified and treated with the second restriction enzyme, DpnII. After a second re-ligation step the sample is purified and a 4C PCR is applied. FACS analysis is used to separate the cells on the basis of the allotypic origin of the productive IgH allele. The FVB allele codes for the B cell receptor from the IgMa isotype, whereas the c57Bl/6 allele encodes for the B cell receptor from the IgMb isotype. Allele specific analysis is enabled applying paired end sequencingon the sorted IgMa and IgMb cell populations. One read (P1) reads the 4C capture, whereas the other read (P2) identifies the genotype of the mouse and thus the productive or non-productive allele.
Illumina adaptor attachment is done by extending the 4C PCR primers with the paired-end Illumina adaptors. These extended 4C PCR primers are used in the 4C PCR protocol
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
4C PCR product
|
| Data processing |
The initial step in the 4C-seq analysis is the alignment of the sequencing reads to a reduced genome of sequences that flank HindIII or DpnII sites (fragment ends), using custom perl scripts. Due to their ambiguous nature in reporting contacts, repetitive fragment ends were excluded from subsequent analysis. Read pairs are used to identify the 4C capture (raw files with .fq extension) and identify the allele by reading the SNP (raw files with .txt extension). The reduced genome was based on mouse mm9.
Genome_build: mm9
Supplementary_files_format_and_content: The processed files are wiggle tracks (variable step), containing the chromosomal location of the fragment-ends present in a HindIII-DpnII digested genome. The first column contains the location per chromosome, the second column contains the number of reads per fragment-end.
|
| |
|
| Submission date |
May 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
sjoerd holwerda |
| E-mail(s) |
s.holwerda@hubrecht.eu
|
| Organization name |
Hubrecht Institute
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE47128 |
Allele specific analysis of the immunoglobulin heavy chain locus by simultaneous analysis of the productive and the non-productive allele through paired-end 4C sequencing analysis in mature B cells |
| GSE47129 |
Allele specific analysis of the immunoglobulin heavy chain locus |
|
| Relations |
| BioSample |
SAMN02152396 |
| SRA |
SRX283907 |
