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| Status |
Public on Jul 13, 2014 |
| Title |
WI-38 CellCycle G Phase rep2 |
| Sample type |
SRA |
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| Source name |
WI-38 cell culture FACS sorted for G phase
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| Organism |
Homo sapiens |
| Characteristics |
cell line: WI-38
cell type: hTERT-immortalized WI-38 primary human lung fibroblasts. Passage 23-27
cell divisions: population
passage: 26
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| Growth protocol |
Undifferentiated human ES cells (hESCs H1/H9) are routinely cultured on mouse embryonic fibroblast feeder layers (MEFs). Single hESC (H1/H9) cells were picked using CellCelector Aviso) using a 30 micron diameter capillary into 96 well plates or 24-well plates. Individual hESC was grown on plate coated with 8x104 iMEFs cells per well and containing conditional media supplemented by hbFGF (8ng/ml, Peprotech) and Rock inhibitor (10μM, Y-27632, Calbiochem). MCF7a and K562 cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10%heat-inactivated fetal calf serum. HepG2 cells was cultured in DMEM medium supplemented with antibiotics and 10%heat-inactivated fetal calf serum. WI-38 primary human embryonic lung fibroblasts were grown in MEM supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics (Biological Industries). WI-38, MCF7 HepG2 and K562 cells were first trypsinized and individual cell was picked in to 96 well plate with condition media. Cells were grown until clonal population was established, 0.05-2X106 cells (2-4 weeks). Naïve T-cells were purified from C57BL/6 splenocytes, cultured in a complete RPMI 1640 medium and were stimulated by IL2 (1.2 ng/ml, R&D system) for two days followed by FACS sorting for CD8+ individual cells by anti CD3 and anti-CD28. Single cell were extended in to clonal population (2.5X104).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from WI-38, hESC (H1, H9), K562, MCF7, HepG2, CD8+ clonal and non-clonal populations (Quick gDNA kit Zymo Research).
gDNA (100ng-300ng ) was digested O.N with MspI (20 units). Size selection was performed, using X0.45 SPRI beads (Agencount AMpure XP, Beckman Coulter, Inc.) in order to remove fragments that are longer than 500bp. The supernatant was separated from the beads and was cleaned by X2.5 SPRI beads in order to select for DNA fragments at size of 200-500bp. Fill in reaction, combined with addition of 3’ overhangs A was performed using Klenow. The product (10ng), was ligated in to 150ng of our T tailed UMI plasmid library and was subjected to bisulfate conversion. The bisulfate conversion protocol was performed based on the manufacturer’s instructions (EZ DNA methylation kit, Zymo Research). Bis-converted fragments were subjected to 20 cycles of amplification with the following thermocycler conditions: 95C for 10 min (95C for 20 s, 56C for 30s, and 72C for 90min), 72 for 5min, using Gotaq hot start polymerase (Promega). In order to amplify and reintroduce C at the XbaI and EcoRI sites we used primers matching the converted sequence but contain C at the right site for site direction mutagenesis. The amplification products were cut by XbaI and EcoRI, subjected to fill in reaction, combined with addition of 3’ overhangs A and were ligated in to modified illumine Truseq adapters followed by 8 cycles of amplification under the same thermocycler conditions that were mentioned above. Products were size selected on 4% 1:3 Nusieve agarose gel (Lonza) for fragments between 200 to 500bp in size and purified using QIAquick gel extraction kit (Qiagen).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
CellCycle_WI38_G_T2.pats
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| Data processing |
We used Bismark (parameters –n 1 –l 20) to map reads to the appropriate genome (hg19). Mapping was done independently for the two ends of each pair. This resulted in 70-80% reads with uniquely defined genomic mapping. We next identified the distance of each mapped read to the nearest MspI site, and discarded all cases that failed to map precisely or within 1bp of such site.
We also discarded read pairs that mapped uniquely to two different fragments. In cases where one read uniquely mapped on a restriction site but the other could not be mapped uniquely or could not be mapped at all, we re-aligned the entire read pair to the fragment.
Given an aligned read pair we first searched for reads with over 1 non-converted cytosine with base quality higher than 20 and discarded them. We then extracted the methylation status of all CpGs in the MspI fragment that were covered by one or both read ends. We defined the sites on the restriction site itself, sites with conflicting methylation status on the two ends, sites with a SNP (A or G), or sites that were not covered as missing data. This resulted in the extraction of a pattern (built from 0, 1 or “*”) from each read-pair.
We discard UMI sequencing errors that are ambiguous (similar to more than one designed UMI with one mismatch). Following this conservative correction, we retain for each restriction fragment (at each strand) at most one pattern for each UMI, by random sampling.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files that include : MspI_Fragment_ID (referenced by MspI_Fragments.coords file) Strand UMI_Barcode Methylation_Pattern Number_of_reads_UMI_got Number_of_stray_barcodes
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| Submission date |
Dec 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Amos Tanay |
| E-mail(s) |
amos.tanay@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Street address |
234 Herzl St.
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| City |
Rehovot |
| State/province |
ISR |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE53610 |
Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells [human] |
| GSE54960 |
Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells |
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| Relations |
| BioSample |
SAMN02485495 |
| SRA |
SRX398357 |
| 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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