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| Status |
Public on Oct 08, 2015 |
| Title |
LC3, Replicate 2 |
| Sample type |
SRA |
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| Source name |
IMR90 fibroblasts in culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
antibody: Cell Signaling Technology #3868
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| Treatment protocol |
No treatment was performed on these cells.
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| Growth protocol |
IMR90 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) under physiological oxygen (3%).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde diluted in PBS, without the addition of other co-crosslinkers, for 5 min at room temperature. After glycin quenching, the cell pellets were lysed in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, 0.1% Na-Deoxycholate, 0.1% SDS, supplemented with complete protease inhibitor cocktail (Thermo Scientific), and sonicated with Covaris sonicator, resulting in chromatin fragments of 250 bp average size. The supernatant was diluted 10 times with the above buffer without SDS, and subjected to immunoprecipitations with 2 ug of antibody or control IgG conjugated with Dynabeads Protein A or G (Invitrogen) at 4 °C for overnight. The beads were then washed 5 times with buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, and 1 time with final wash buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 50 mM NaCl), followed by elution with incubation of elution buffer (final wash buffer plus 1% SDS) at 65 °C for 30 min with agitation in a thermomixer.
The ChIP and input were purified and used for qPCR analysis or for constructing sequencing libraries with the NEBNext Ultra kit (New England Biolabs).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Demultiplexing of NextSeq data was performed using bcl2fastq v2.16.0.10 with default parameters.
Data were aligned to the GRCh37 (hg19) assembly of the human genome using bowtie2 with command-line parameters -k1 -N1 --local (allowing and reporting a single alignment per read with one or zero mismatches permitted in the seed region).
ChIP-seq visualization tracks were created in the following way. Aligned sequence tags were subjected to bedtools' genomeCoverageBed tool, making bedGraphs that were multiplied by the RPM coefficient. A similarly normalized input bedGraph was then subtracted from Lamin B1 and LC3, and bedGraphs were made into bigWigs using UCSC Genome Browser's bedGraphToBigWig utility.
Enriched domains for Lamin B1 and LC3 were called using EDD with default bin size estimation and gap penalty estimation and unalignable regions (the hg19 assembly gap track from Genome Reference Consortium) were masked. The FDR was controlled at the default value of 5%.
Genome_build: hg19
Supplementary_files_format_and_content: Tracks (bigWig files) were created by running genomeCoverageBed from the bedtools software package to create bedGraphs and adjusting tag counts by the RPM coefficient for each replicate of lamin B1, LC3, and input. Input bedGraphs were then subtracted from lamin B1 and LC3 bedGraphs and these were converted to bigWigs using bedGraphToBigWig from the UCSC Genome Browser utilities. BED files describing large domains of lamin B1 or LC3 enrichment were obtained by running EDD using default bin size estimation and gap penalty estimation. Unalignable regions (gaps) were masked and the FDR was controlled at 5%.
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| Submission date |
Jul 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
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| Department |
Cell & Developmental Biology
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| Lab |
Zaret Lab
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE63440 |
Autophagy mediates degradation of nuclear lamina |
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| Relations |
| BioSample |
SAMN03890912 |
| SRA |
SRX1112996 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1827556_LC3-Input.R2.bw |
889.3 Mb |
(ftp)(http) |
BW |
| GSM1827556_LC3ADs.R2.EDD.bed.gz |
3.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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