 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 19, 2020 |
| Title |
mLANA_ChIP_3A_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
BALB/c B cell lymphoma
|
| Organism |
Mus musculus |
| Characteristics |
cell line: A20-mLANA 3A
modification to cells: A20 cells transduced with lentivirus containing FLAG-tagged mLANA 3A IRES-EGFP expression construct
treatment: Dox
chip antibody: EZview Red anti-FLAG M2 Affinity Gel (Sigma-Aldrich F2426)
|
| Treatment protocol |
BCBL-1, BJAB-mCherry, BJAB-kLANA and BJAB-rKSHV.219 cells were plated in T-75 flasks at a cell density of 0.5 x 10^6 cells/ml and in the absence or presence of I-BET151 at a final concentration of 0.5 µM for 48 hours. For induction of A20-GFP, A20-mLANA WT or A20-mLANA 3A, cells were cultured in a T-75 flask at a density of 0.2 x 10^6 cells/ml supplemented with doxycycline at a final concentration of 1 µg/ml.
|
| Growth protocol |
BCBL-1, BJAB-mCherry, BJAB-kLANA and BJAB-rKSHV.219 were cultured in RPMI 1640 (Gibco) with 20% (v/v) Foetal Calf Serum (FCS) (HyClone) whereas A20-GFP, A20-mLANA WT and A20-mLANA 3A mutant were cultured in RPMI 1640 with 10% (v/v) FCS. All cells were incubated in a humidified incubator with 5% CO2 at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described in (Si, Verma, & Robertson, 2006; Günther & Grundhoff, 2010) with the following modifications. For kLANA and mLANA ChIP, the formaldehyde fixation time was 10 minutes, while for Brd2/4 it was 20 minutes. For the Brd2/4 ChIP experiments we used rabbit polyclonal antigen-affinity purified IgG antibodies (Bethyl laboratories, Inc. BRD2-A302-583A and BRD4-A301-958A100) at a concentration of 3 µg/mg of lysate. Antibodies were incubated with the lysates for 6 hours before adding protein G beads and then further incubated for 16 hours. For kLANA ChIP, we used an IgG2c rat monoclonal antibody (Advanced Biotechnologies Inc. 13-210-100) and prepared antibody-conjugated beads as previously described (Zhang et al., 2016), with the exception of the beads being washed and finally resuspended in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris HCl - pH 8.0, 167 mM NaCl). Rabbit IgG Cell Signaling Technology® and Rat IgG Abcam were used as isotype controls in, respectively, the Brd2/4 and kLANA ChIP experiments. For the mLANA experiments, we used 50 µl of EZview™ Red anti-FLAG® M2 Affinity Gel (Sigma-Aldrich) per lysate derived from ~3 x 10^7 nuclei.
Libraries were prepared using the NEXTflex™ ChIP-Seq and NEXTflex™ ChIP-Seq Barcodes - 12 kits from BIOO Scientific Corp. according to the manufacturer’s instructions (NEXTflex™ ChIP-Seq kit Manual 5143-02) to prepare single-end DNA libraries from ChIP DNA using the gel-free protocol and libraries were size selected to be between 300-400 bp.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basecalling was performed using the built-in Real-Time Analysis (RTA) software version 2.4.11.
All FASTQ sequence files were checked for quality using the FastQC tool. Reads exceeding 51 bp were trimmed from the 3’ end down to 51 bp using the FASTQ/A trimmer tool from the FASTX-toolkit.
We used Bowtie for aligning reads to the mouse (mm9) genome for mouse datasets, and human (hg19) genome for human datasets, using the default options with the following exceptions: --best, --strata and -m 1. For data from cells containing the KSHV genome, reads were also aligned to the HQ404500 sequence to which two copies of TRs were appended. Alignment of the reads to the long unique region (LUR) was performed with the aforementioned settings. All resulting SAM files were sorted, duplicates were removed and finally converted to BAM format.
Peak calling was performed using MACS using the default options with the following exceptions: --nomodel and --shiftsize value was set to half the size of mean fragment length of the ChIP-Seq library as determined by Agilent 2100 Bioanalyzer. For KSHV LUR, peak calling was performed with the aforementioned settings but with the genome size set to 137706 bp and using an additional option --nolambda. For alignment to the terminal repeat (TR) regions of the KSHV genome, reads were aligned using Bowtie with the aforementioned options for read alignment, except -m 1 was changed to -M 1. The SAM files were sorted and converted to BAM without removal of duplicates. No peak calling was performed for alignments for the TR. Instead, the aligned reads were extended to match the library fragment size using the igvtools ‘Count’ command from the Integrative Genomics Viewer (v2.3) with default settings, the window size set to 10 and the extension factor set depending on the average fragment size of the library.
Genome_build: hg19 (GRCh37) for human genome, HQ404500 appended with two copies of TRs for KSHV genome and mm9 (MGSCv37) for mouse genome.
Supplementary_files_format_and_content: wig files were generated during peak calling step using the MACS peak caller.
|
| |
|
| Submission date |
Jun 25, 2020 |
| Last update date |
Oct 19, 2020 |
| Contact name |
Rishikesh Lotke |
| Organization name |
Medizinische Hochschule Hannover
|
| Department |
Institut für Virologie
|
| Lab |
Thomas F. Schulz
|
| Street address |
Carl-Neuberg-Straße 1
|
| City |
Hannover |
| State/province |
Niedersachsen |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE153243 |
Brd/BET Proteins Influence the Genome-Wide Localization of the Kaposi’s Sarcoma-associated Herpesvirus and Murine Gammaherpesvirus Major Latency Proteins [ChIP-seq] |
| GSE153244 |
Brd/BET Proteins Influence the Genome-Wide Localization of the Kaposi’s Sarcoma-associated Herpesvirus and Murine Gammaherpesvirus Major Latency Proteins |
|
| Relations |
| BioSample |
SAMN15368131 |
| SRA |
SRX8616384 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4636657_mLANA_3A_vs_A20_rep2_mm9_macs_treat_afterfiting_all.wig.gz |
381.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
