Illumina HiSeq 2000 paired end sequencing; BAR-ChIP on yeast chromatin associated mutants
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Yeast strain
Cell type
BY4741
NA
NA
Attributes by original data submitter
Sample
ENA first public
2015-01-15
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA2779903
INSDC center alias
European Molecular Biology Laboratory
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2015-01-15T17:01:43Z
INSDC last update
2018-03-08T21:09:37Z
INSDC status
public
Submitter Id
E-MTAB-2945:WCE_delta_rco1_3
broker name
ArrayExpress
common name
baker's yeast
genetic modification
gene knock out
genotype
BY4741_delta_rco1
sample name
E-MTAB-2945:WCE_delta_rco1_3
sex
mating type a
strain
BY4741
Sequenced DNA Library
library_name
WCE_delta_rco1_3
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown in 50 mL YPD (1% yeast extract, 2% peptone, 2% glucose and 40 mg adenine) and harvested at an OD600 of ~1 Cross-linking was performed with 1% formaldehyde (final concentration) for 15 min at room temperature on a shaker plate. Fixation was quenched with the addition of Glycine to a final concentration of 0.135M. Cross-linked cells were washed three times in ice-cold TBS buffer (20 mM Tris pH 7.5, 0.146 M NaCl). Yeast pellets were resuspended in NP-S buffer (0.5 mM spermidine, 0.075% NP40, 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 1mM ??-mercaptoethanol) with Protease Inhibitor Cocktail (Roche) and lysed at 4??C with 2 volumes of glass beads in a FastPrep instrument (MP Biomedicals) using the following settings: 4 x 20 sec at 6.5 m/sec with 1 min pause between each pulse. Nucleosome fractions were isolated by incubating lysates with 80U of Micrococcal Nuclease (Worthington Biochemicals) for 45 min at 37??C. EGTA was added to samples at a final concentration of 0.01M for enzyme inactivation. Barcoding of nucleosome fractions was performed using 50 ??g of starting material as measured by absorbance at 260nm with Nanodrop. Chromatin fragments were end-repaired with 2 ??L of NEBNext End repair Enzyme Mix (New England BioLabs), dA-tailed with 2 mM dATP and 20U Klenow Fragment (Thermo Scientific) and ligated for 4 hrs at 16??C to paired-end adapters containing a 6-mer multiplex barcode with 1600U T4-DNA Ligase (New England BioLabs). Enzymes were heat-inactivated at 65??C-70??C between each step. Antibodies were diluted in PBS containing 5 mg/mL BSA (Sigma) and 10-30 microg of yeast tRNA (Ambion) and conjugated to 1.5 mg and 4.5 mg of protein A Dynabeads (Invitrogen) for classical ChIP and HT-ChIP respectively, for 4 hrs at 4C on a wheel. 50 microg of non-barcoded nucleosomes (classical ChIP) were diluted in lysis buffer (50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1 % Sodium Deoxycholate, 1 mM Benzamidin and Protease Inhibitor Cocktail) and incubated with antibody-coated magnetic beads overnight at 4C on a wheel. Chromatin-antibody complexes were washed 8 times in RIPA buffer (0.05 M Hepes, 0.5 M LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Sodium Deoxycholate) and chromatin was eluted from the antibody-coated beads by two 10 min-incubations in ChIP elution buffer (50 mM Tris pH 8.0, 10 mM EDTA and 1% SDS) at 68C with mixing. Formaldehyde crosslinking, Cell lysis using Fast-prep, Micrococcal nuclease digestion, Chromatin IP, Reverse crosslinking and library preparation 20 to 100 ng of IP- and input DNA were end-repaired, dA-tailed and ligated to paired-end adapters as described above. Purification of the newly barcoded DNA samples was performed using Ampure XP beads. 1 ng of barcoded IP- and input DNA from BAR- and classical ChIP experiments was amplified by PCR using 0.4 ??L of each Illumina Paired-End primers (at 10 ??M) and 20 ??L of 2X Phusion HF Master Mix (New England Biolabs) with the following program: 30 sec at 98 ??C for initial denaturation, 30 sec at 98??C, 30 sec at 65??C, 30 sec at 72??C for 18 cycles, followed by 5 min at 72??C for final extension