Lysates were prepared by bead beating in FA lysis buffer (50mM HEPES pH7.5, 1mM EDTA, 1% Triton X-100, 0.1%sodium deoxycholate and 150mM NaCl). The ChIP lysates were used to immunoprecipitate acetylated H4 and histone H3. The immune complexes were pulled down with 50 ml of Protein G Dynabeads (Invitrogen) per IP and washed with FA lysis buffer, FA lysis buffer with 1M NaCl, FA lysis buffer with 0.5M NaCl, TEL buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCL, pH8.1), and twice with TE. The Immune complexes were eluted with Elution buffer (1%SDS in TE with250mM NaCl). The eluates were RNase treated and Proteinase K was added to remove the bound proteins. Crosslinkss were reversed by incubating at 65 C overnight. Samples werte phenol:chloroform treated and ethanol precipitated. Input samples were treated the same way from the RNase step.
Label
cy5
Label protocol
Briefly, up to 50 ng of IP or input DNA was treated with 2.5 U CIP enzyme (NEB) for 1 hour at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37°C for T-tailing the ends, and the product isolated with the MinElute Reaction Cleanup Kit (Qiagen). An anchored T7 promoter-(dA)18 oligo was added by annealing and filling with 5 U Exo- Klenow (NEB) for 4 hours at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37°C overnight using the Ampliscribe T7 transcription kit (Epicentre Biotechnologies). RNAs purified using the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). For the second round amplification, 100–150 ng of RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). The T7 promoter primer was added to the product through a Klenow filling reaction as described above, followed by in vitro transcription with amino allyl-UTP (Ambion) instead of UTP. Final reaction cleanup was performed with the RNeasy Mini Kit (Qiagen). For the labeling reactions, 8 ug of amino allyl-incorporated RNA in a 5 uL volume of 0.1 M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) Cy3 or Cy5 dye (GE Healthcare) in DMSO (Sigma) and incubated at room temperature for 2 hours. Reactions were quenched with 5 uL 4 M hydroxylamine at room temperature for 15 minutes, purified by RNeasy MinElute Cleanup Kit (Qiagen), and the dye incorporation measured using the Nanodrop 2000 (Thermo Scientific). 2.5-4µgg of input and IP were combined and fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions.
Lysates were prepared by bead beating in FA lysis buffer (50mM HEPES pH7.5, 1mM EDTA, 1% Triton X-100, 0.1%sodium deoxycholate and 150mM NaCl). The ChIP lysates were used to immunoprecipitate acetylated H4 and histone H3. The immune complexes were pulled down with 50 ml of Protein G Dynabeads (Invitrogen) per IP and washed with FA lysis buffer, FA lysis buffer with 1M NaCl, FA lysis buffer with 0.5M NaCl, TEL buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCL, pH8.1), and twice with TE. The Immune complexes were eluted with Elution buffer (1%SDS in TE with250mM NaCl). The eluates were RNase treated and Proteinase K was added to remove the bound proteins. Crosslinkss were reversed by incubating at 65 C overnight. Samples werte phenol:chloroform treated and ethanol precipitated. Input samples were treated the same way from the RNase step.
Label
cy3
Label protocol
Briefly, up to 50 ng of IP or input DNA was treated with 2.5 U CIP enzyme (NEB) for 1 hour at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37°C for T-tailing the ends, and the product isolated with the MinElute Reaction Cleanup Kit (Qiagen). An anchored T7 promoter-(dA)18 oligo was added by annealing and filling with 5 U Exo- Klenow (NEB) for 4 hours at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37°C overnight using the Ampliscribe T7 transcription kit (Epicentre Biotechnologies). RNAs purified using the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). For the second round amplification, 100–150 ng of RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). The T7 promoter primer was added to the product through a Klenow filling reaction as described above, followed by in vitro transcription with amino allyl-UTP (Ambion) instead of UTP. Final reaction cleanup was performed with the RNeasy Mini Kit (Qiagen). For the labeling reactions, 8 ug of amino allyl-incorporated RNA in a 5 uL volume of 0.1 M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) Cy3 or Cy5 dye (GE Healthcare) in DMSO (Sigma) and incubated at room temperature for 2 hours. Reactions were quenched with 5 uL 4 M hydroxylamine at room temperature for 15 minutes, purified by RNeasy MinElute Cleanup Kit (Qiagen), and the dye incorporation measured using the Nanodrop 2000 (Thermo Scientific). 2.5-4µgg of input and IP were combined and fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions.
Hybridization protocol
The hybridization mixture (Oligo CGH/Chip-on-chip hybridization kit, Agilent) was prepared according to manufacturer’s instructions with the addition of 20 ug of T7 blocking oligo (ttt ttt ttt ttt tt cac gcg tct ccc) , and hybridized overnight at 65°C. Microarrays were washed using the Oligo aCCH/Chip-on-chip Wash buffers (Agilent) according to manufacturer’s instructions.
Scan protocol
Scanned on Agilent Technologies Scanner G2505C US45102919 Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
Description
Biological Replicate 2 of 3. ChIP enrichment of AcH4 IP over Input in the ASF1 BY4741 strain.
