|
| Status |
Public on Feb 16, 2018 |
| Title |
FXS iPSC_dC-dT_ChIP-BS-seq |
| Sample type |
SRA |
| |
|
| Source name |
FXS iPSCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: FXS iPSC line containing about 450 CGG repeats in the 5' UTR region of FMR1
genotype: Infected with catalytically inactive Tet1 (dC-dT) and sgRNA targeting CGG repeats
chip antibody: Mouse monoclonal anti-Cas9
|
| Treatment protocol |
Cells were cross-linked by 1% formaldehyde in the medium for 10 min in room temperature, and then quenched by adding 0.125 M Glycine for 5 min.
|
| Growth protocol |
FXS iPSCs were cultured either with mTeSR™1 medium (STEMCELL, #85850) or on irradiated mouse embryonic fibroblasts (MEFs) with standard hESCs medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Collected cells were washed with PBS twice, and then re-suspended in 3.5 ml of sonication buffer. Sonication was performed for 10 cycles with 0.5 min pulse on and 1 min rest, and 24 watts in ice-water mixture. Then cell lysate was spun down with 14,000 x rpm for 10 min at 4 °C. 50 ul of supernatant was saved as input for gDNA. 10 ul of anti-CTCF antibody (EMD Millipore: 07729) was added and incubate overnight at 4 °C. 50 ul protein G dynabeads was added into antibody-cell lysate mixture and incubate overnight at 4 °C. Then beads were washed with sonication buffer, sonication buffer with high salt (500 mM NaCl), LiCl wash buffer, and TE buffer. Bound protein-DNA complex was eluted from beads by incubation in a 65 °C oven for 15 min, and then reverse cross-linked under 65 °C over-night. The bound DNA was purified with Qiagen QIAquick PCR Purification Kit.
EpiNext High-Sensitivity Bisulfite-Seq Kit.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Chip-Seq then Bisulfite conversion
|
| Data processing |
The adapter sequences in the Illumina reads identified with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) were removed with Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). BS-Seq aligner Bismark was used for assigning reads to human genome hg19 and calling methylation with bismark_methylation_extractor. To increase the number of uniquely mapped reads, after the first bismark alignment, 5 bases from the 5’ and one base from the 3’ of the unmapped reads were trimmed based on FastQC analysis. The resulting trimmed reads were then aligned to genome with bismark. In both cases, bismark was ran with the options “--non_directional -un --ambiguous --bowtie2 -N 1 -p 4 --score_min L,-6,-0.3 --solexa1.3-quals”.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *_sorted.bismark.cov: Tab-delimited text files from bismark: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count unmethylated>
|
| |
|
| Submission date |
Dec 17, 2017 |
| Last update date |
Feb 18, 2018 |
| Contact name |
Shawn Liu |
| E-mail(s) |
shawnliu@wi.mit.edu
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Rudolf Jaenisch
|
| Street address |
455 MAIN STREET
|
| City |
CAMBRIDGE |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE108171 |
Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene [methylation] |
| GSE108577 |
Rescue of Fragile X syndrome by DNA methylation editing of the FMR1 |
|
| Relations |
| BioSample |
SAMN08195259 |
| SRA |
SRX3477559 |
