|
| Status |
Public on Apr 06, 2017 |
| Title |
Tip60fl/+_Tip60ciRNAseq_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
murine embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129/Ola
passage: 13-16
treatment: untreated
genotype/variation: Tip60fl/+ ES cells
cell line: E14
|
| Treatment protocol |
Lentivirus sepcific to Tip60 is used for Tip60KD.
|
| Growth protocol |
E14 mouse embryonic stem cells (ESCs) were cultured on gelatin-coated dishes in high-glucose DMEM medium (Sigma) containing 10% serum (Corning) and LIF at 37°C with 5% CO2. During differentiation, ESCs were cultured without LIF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using TRIzol and further purified on RNA Clean and Concentrator columns for RNAseq
For ATACseq, cells were treated with lysis buffer containing non-ionic detergent to generate nuclei
Two independent ATAC reactions per biological replicate were performed, using 35,000 and 70,000 ESCs each. After library preparation (Buenrostro:2013, Buenrostro:2015), the two reactions were found to have indistinguishable distributions of fragment sizes, and were therefore combined for sequencing. (Therefore, each biological replicate consisted of two ATAC reactions).
Strand specific library construction and RNAseq were performed by Applied Biological Materials, Inc. and the UCLA Clinical Microarray Core for ESCs and differentiating ESCs, respectively
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
TopHat2 was used to map the RNAseq reads to the mouse genome (mm10). The bam files from the Tophat output were used for downstream analysis using HOMER. DESeq2 was used to identify the differentially expressed genes. Heatmaps were generated using Java TreeView. K-means clustering was performed using Cluster 3.0.
For ATACseq: Paired-end 75 bp reads were trimmed to 24 bases and reads were then aligned to mm10 using Bowtie2 with the parameter -X 2000 to ensure that fragments up to 2 kb were allowed to align. Duplicates were then removed using Picard. Reads with low quality score (MAPQ < 10) and reads mapping to the mitochondrial genome were removed. Reads were separated into size classes, and only nucleosome free reads (less than 100 bp) were used for subsequent analyses. These reads were processed in HOMER. Genome browser tracks were generated from mapped reads using the “makeUCSCfile” command. Peaks were individually called from replicate datasets using the “findPeaks” command and those peaks enriched in both replicates were retained. Mapped reads were aligned over specific regions using the “annotatePeaks” command to make 20 bp bins over regions of interest and sum the reads within each bin. Experiments were aligned over the following datasets: TSS reference sites (from HOMER software), DNaseI hypersensitive sites (DHSs) from mouse ENCODE data (GSM1014154) with TSS locations removed, and high quality (peak score > 6) Tip60 peaks called from (Raven et al 2015). After anchoring mapped reads over reference sites, aggregation plots were generated by averaging data obtained from biological replicates.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph
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| |
|
| Submission date |
Aug 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Thomas Fazzio |
| E-mail(s) |
Thomas.Fazzio@umassmed.edu
|
| Organization name |
UMMS
|
| Street address |
364 Plantation street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL21493 |
| Series (1) |
| GSE85505 |
KAT–independent gene regulation by Tip60 promotes ESC self-renewal but not pluripotency |
|
| Relations |
| BioSample |
SAMN05560741 |
| SRA |
SRX2013621 |
