DNA extraction and shearing procedures were performed as indicated previously32 (link), 33 (link). ChIP was carried out with SX-8G IP-Star® Compact Automated System (Diagenode). Anti-H3K27me3 (pAb-069-050), anti-H3K9ac (pAb-103-050) and non-immune rabbit IgG (Kch-504-250) were obtained from Diagenode. The performance of ChIP experiments was confirmed by Q-PCR (ABI PRISM 7900HT, Applied Biosystems). Primer sequences of human gene TSH2B (Diagenode) were used as positive control for methylation and C-FOS (Diagenode) for acetylation. The experimental protocol was conducted according to32 (link), 33 (link).
Sx 8g ip star compact automated system
Manufactured by Diagenode
The SX-8G IP-Star® Compact Automated System is a lab equipment product designed for automated DNA and RNA extraction and purification. It features a compact design and can process up to 8 samples simultaneously.
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5 protocols using sx 8g ip star compact automated system
1
Chromatin Immunoprecipitation and Quantitative PCR
2
SATB1 Chromatin Immunoprecipitation Protocol
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DNA bound proteins were cross linked to DNA by exposing cells to 1 % PFA for 10 min at RT. Cross linking was quenched with 0.25 M glycine. Cell nuclei were extracted and lysed with 3 different buffers used in a sequential order. Buffers contained 50 mM HEPES-KOH, 140 mM NaC, 1 mM EDTA, 10 % glycerol, 0.5 % NP-40, 0.25 % Triton X-100 (Buffer 1); 200 mM NaCl, 1 mM EDTA 0.5 mM EGTA, 10 mM Tris pH 8.0 (Buffer 2); 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8.0, 100 mM NaCl, 0.1 % Na-Deoxylcholate and 2.5 mL N-lauroyl sarcosines. Chromatin was sheared 8 times for 30 s with 30 s breaks at 4C in Buffer 3 with a CHIP grade sonicator (Diagenode). Chromatin immunoprecipitation and subsequental DNA extraction was performed with the Auto Transcription ChIP kit and Auto IPure kit using the SX-8G IP-Star® Compact Automated System (all Diagenode) following the manufacturers protocol. The extent of SATB1 bound DNA was measured by PCR comparing the eluate of the chromatin immunoprecipitated sample to its corresponding input sample.
3
ChIPmentation Protocol for Transcription Factor
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ChIPmentation was performed using a ChIPmentation for Transcription Factor kit (Diagenode) according to the manufacturer’s instructions. Briefly, the cells were fixed with 1% formaldehyde for 8 min. Chromatin was sheared by sonication using a Picoruptor® (Diagenode) for ten cycles and subjected to magnetic immunoprecipitation and tagmentation using an SX-8G IP-STAR® Compact Automated System (Diagenode). The sequencing libraries were amplified by nine cycles of PCR and cleaned up using AMPure XP beads (Beckman Coulter). The ChIPmentation libraries were sequenced using 150 bp paired-end reads on the HiSeq X (Illumina). The sequence reads were mapped to the human hg19 genome by bowtie2. Reads mapped to the mitochondrial genome and duplicated reads were removed using removeChrom.py and samtools, respectively. Peak calling was performed using macs2 with a 10−10 cutoff P value. The antibody used for the ChIPmentation were shown in Supplementary Table 2 . The ChIPmentation was performed in two biological replicates.
4
ER and KDM3A Chromatin Enrichment
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Chromatin was harvested from MCF-7 and T47D cells and fractionated by sonication as described (19 (link)). ChIP was performed using the Auto ChIP protein A kit (Diagenode) in the SX-8G IP-Star Compact Automated System (Diagenode) following manufacturer's instructions. Briefly, 30 μg of DNA and 2 μg of ER antibody, 3 μg of KDM3A antibody, 1 μg of H3K9me1/2 antibodies and equal amounts of isotype control antibodies were used for each ChIP reaction which comprised of 2-h antibody coating and 10-h immunoprecipitation incubation periods (antibody details: Supplementary Table S2). Following ChIP, eluted DNA and input samples taken from the original sonicated sample were subject to cross-link reversal and qPCR using primers specific to pS2 and GREB1 promoter elements and CCND1, MYC and XBP1 distal enhancer elements (primer sequences: Supplementary Table S4). Data were calculated as % input (as described in (19 (link))). Data were presented as the average fold difference of % input between different experimental arms (detailed in figure legends) of at least three independent experiments.
5
Automated ChIP-qPCR for Histone Modifications
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Chromatin immunoprecipitation was performed using an AUTO True MicroChIP KIT (Diagenode) according to the manufacturer's protocol on an SX-8G IP-Star® Compact Automated System (Diagenode). ChIP used 200 μL of sonicated chromatin and 3 μg of antibodies: anti-H3K27me3 (#C15410069, Diagenode), anti-EZH2 (#C15410039, Diagenode), anti-JMJD3 (#ab85392, Abcam) and anti-IgG for negative control (#C15410206, Diagenode). Antibody coating reaction with protein A-coated magnetic beads lasted 3 h, and the immunoprecipitation reaction 13 h at 4° C. Reverse cross-linking was carried out for 4 h at 65° C.
Immunoprecipitated DNA (IP) and total DNA (input) were purified by MicroChIP DiaPure columns (#C03040001, Diagenode) according to the manufacturer's instructions, and analyzed by real-time PCR.
Immunoprecipitated DNA (IP) and total DNA (input) were purified by MicroChIP DiaPure columns (#C03040001, Diagenode) according to the manufacturer's instructions, and analyzed by real-time PCR.
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