LowCell# ChIP kit protein A x16

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.	-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. - Chromatin from 1,000 cells is sufficient for one ChIP assay.	-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Upstream tips
-After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.
Protocol tips
-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. - Chromatin from 1,000 cells is sufficient for one ChIP assay.
Downstream tips
-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Publication protocol

Around twelve million cells were cultured separately from the RNA-seq experiments but in identical conditions as previously mentioned (37 °C, 5% CO2 for two passages) on 6 well plates. For each immunoprecipitation, one plate was fixed with 1% formaldehyde for 15 minutes and quenched with 0.125 M glycine. The remainder of the ChIP-seq protocol was carried out using the Diagenode LowCell# ChIP kit (Diagenode; catalog number: C01010070) following the manufacturer’s protocol. Chromatin was isolated by adding lysis buffer and sheared to an average length of 300 bp with a Covaris sonicator. Genomic DNA regions of interest were isolated using 4 ug of antibody against H3K27ac (Abcam; catalog number ab4729). Genomic DNA complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation at 65 °C, and ChIP DNA was subsequently isolated and purified. Three technical replicates were carried out for each condition. Input genomic DNA was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a Bioanalyzer. ChIP and input DNAs were prepared for amplification using a ThruPLEX DNA-seq kit (Rubicon Genomics) following the manufacturer’s protocol. Library barcode adaptors were added to each sample during amplification and the library was size-selected (~150–200 bp) using a Diagenode iPure kit v2. The resulting amplified DNA was purified, quantified, and tested by a Bioanalyzer reading to assess the quality of the amplification reactions. Amplified DNA libraries were sequenced on and Illumina HiSeq. 4000. All reads were mapped to the human genome using Bowtie51. H3K27ac peaks were called against input using MACS52 and peaks consistent across replicates identified using the ENCODE Irreproducibility Discovery Rate (IDR) pipeline53. For differential peak intensity analysis, peaks across conditions were merged and reads coverage obtained using HTSeq.54. H3K27ac peaks differentially enriched in the PenStrep condition were then identified through DESeq. 29 using a custom normalization matrix to correct for differing signal to noise ratios between replicates55. The mean number of normalized counts per region was calculated across all replicates in both conditions. The difference between the mean and the normalized counts per regions (corrected for signal to noise ratios) was used as a z-score for visualizing differential H3K27ac peak enrichment. As with the RNA-seq analysis, PenStrep dependent regions were identified through clustering analysis on all differentially enriched H3K27ac regions with adjusted p-values (FDR) < 0.1 between the PenStrep-treated and non-treated conditions using the R package hclust and displayed in a heatmap.

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Manufacturer protocol

Download the product protocol from Diagenode for LowCell# ChIP kit protein A x16 below.

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