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| Status |
Public on Sep 11, 2012 |
| Title |
ChIP-Seq of Rela 60 min post LPS stimulation |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell type: bone marrow-derived dendritic cells
strain: C57BL/6
chip antibody: Rela
treatment: LPS
time: 60 min
chip antibody manufacturer 1: Bethyl
chip antibody catalog number 1: A301-824A
chip antibody manufacturer 2: Santa Cruz
chip antibody catalog number 2: sc-372
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| Growth protocol |
Briefly, 6-8 week old female C57BL/6J mice were obtained from the Jackson Laboratories. RPMI medium (Invitrogen) supplemented with 10% heat inactivated FBS (Invitrogen), β-mercaptoethanol (50uM, Invitrogen), L-glutamine (2mM, VWR) penicillin/streptomycin (100U/ml, VWR), MEM non-essential amino acids (1X, VWR), HEPES (10mM, VWR), sodium pyruvate (1mM, VWR), and GM-CSF (20 ng/ml; Peprotech, Rocky Hill, NJ) was used throughout the study. At day 0, bone marrow-derived dendritic cells (BMDCs) were collected from femora and tibiae and plated on twenty (per mouse), 100mm non-tissue culture treated plastic dishes using 10ml medium per plate. At day 2, cells were fed with another 10ml medium per dish. At day 5, cells were harvested from 15ml of the supernatant by spinning at 1400rpm for 5 minutes; pellets were resuspended with 5ml medium and added back to the original dish. Cells were fed with another 5ml medium at day 7. At Day 8, all non-adherent and loosely bound cells were collected and harvested by centrifugation. Cells were then resuspended with medium, plated at a concentration of 15x106 cells in 10ml medium per 100mm dish. At day 9, cells were stimulated for various time points with LPS (100ng/ml, rough, ultra-pure E. coli K12 strain, Invitrogen).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Solid-phase reversible immobilization (SPRI) cleanup steps were performed using the Bravo liquid handling platform (Agilent) using a modified version of (Fisher et al., 2011). 120ul SPRI AMPure XP beads (Agencourt) were added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in 40 ul EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction products (without moving them from their original well position). After thorough mixing and a 2-minute incubation at room temperature, plates are transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are then washed on the magnet with 150ul 70% ethanol and then air dried for 4 minutes. The DNA is eluted with 40ul of EB buffer by pipette mixing 25 times. Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27 µl of a master mix (17 µl master mix (5 ul T4 buffer, 5ul BSA-1mg/ml, 5ul ATP-10mM -2ul dNTPs 10 mM), 5 ul T4 PNK enzyme, 5 µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12C for 15 min, 25C for 15 min, and finally cooled to 4C. The SPRI bead clean up method was used to purify the product (147 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). The A-base addition was performed by adding 20 µl master mix (17 µl A-base add mix, 3 µl Klenow (3’->5’ exonuclease) to each well and incubated at 37C for 30 min. in a thermal cycler. SPRI bead clean up method was used to purify the product (132 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 19 µl EB). Adaptor ligation was performed by adding 34 µl of a master mix (29 µl 2x DNA ligase buffer, 5 µl DNA ligase) to each well. Finally 5 µl PE Indexed oligo adaptors (0.75 uM ) was added to each well and samples were incubated 25C for 15 min in a thermal cycler. SPRI bead clean up with size selection was used to purify the ligated products (15.5 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). Finally, enrichment PCR was performed by adding 10 µl of a master mix (2 µl Forward/Reverse Index Primer, 0.5 µl dNTP mix, 5 µl 10x Pfu Ultra Buffer, 1 µl Pfu Ultra-II Fusion, 1.5 µl Nuclease free water) to each well. Plate was transferred to a thermal cycler and ran a Pfu amplification program at 95C for 2 min, 16 cycles of: 95C for 30 sec, 55C for 30 sec, 72C for 60 sec, and finally 72C for 10 min. The final SPRI clean up coupled to size selection was performed (35 µl SPRI beads was added to each sample and eluted in 40 µl). Sample concentrations were measured and 5 µl was used for ChIP-String enrichment validation.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
Peaks were called using the ChIP-Seq module of the Scripture segmentation program (http://www.broadinstitute.org/software/scripture/)
Genome Build: mm9
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| Submission date |
Feb 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Manuel Garber |
| Organization name |
University of Massachusetts Medical School
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| Department |
Bioinformatics and Integrative Biology
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| Lab |
Garber
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| Street address |
55 Lake Ave North Worcester
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| City |
Worcester |
| State/province |
Massachusetts |
| ZIP/Postal code |
01655 |
| Country |
USA |
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| Platform ID |
GPL15103 |
| Series (2) |
| GSE36099 |
A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals (ChIP-Seq) |
| GSE36104 |
A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals |
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| Relations |
| SRA |
SRX122471 |
| BioSample |
SAMN00792564 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM881114_Rela_60.bw |
998.8 Mb |
(ftp)(http) |
BW |
| GSM881114_Rela_60_peaks.txt.gz |
977.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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