Anyone using Bioruptor Pico?

[dbrg77]

Hi,

I'm wondering whether anyone here has ever succeeded using Bioruptor Pico sonicator for transcription factor ChIP-qPCR.

Before, we had an old standard Bioruptor which worked okay with both ChIP-qPCR and ChIP-seq. Recently, we've switched to the new product from Diagenode, i.e. Bioruptor Pico. When we look at the input sample on agarose gel, it suggests that the new sonicator is very efficient for chromatin shearing (if using their special tubes from Diagenode).

However, I am currently unable to perform a successful ChIP-qPCR (no enrichment, Ct of ChIP samples is very very high).

I'm sure both the protocol and antibodies are fine, as we have been doing ChIP-qPCR and ChIP-seq for years without any problem using the same procedure, except, of course, the sonicator.

I ran my input sample and IPs on western blot, the strange thing is that I cannot even detect my TF in the input sample ......

I am wondering whether anybody here has the same problem, or anybody is using this sonicator without any problems.

Thank you.

[richi17]

Hi,
We were able to generate nice ChIP-seq data comparable to ENCODE using CTCF, p300 and LSD1. We have noticed that ChIP recovery (% of input) drops slightly on chromatin shared on Pico but at the same time the fold enrichment (ratio pos/neg loci) is usually higher compared to chromatin sheared on old Bioruptors.
Comparing to an old Biorupotrs, Bioruptor Pico is a way more performant. Consequently, a sonication protocol should be re-adapted to Pico by decreasing a sonication time. Usually, 5-10 cycles are already enough for an efficient shearing with fragments in the range 100-300 bp (an ideal range for ChIP-seq) . In some cells type, even shorter sonication time (2-3 cycles) resulted in a nice shearing. It is important to perform a pilot experiment before ChIP to figure out how long to sonicate. The golden rule is always to choose the shortest sonication. For some antibodies, it is worth trying a longer fixation (15-20 min). Regarding ChIP-qPCR, primers sets should be revised and primers amplifying short PCR products (80-100 bp) should be preferred. Longer PCR products could lead to reduced sensitivity in qPCR.

Diagenode-Bioruptor Pico

[dbrg77]

Hi,
We were able to generate nice ChIP-seq data comparable to ENCODE using CTCF, p300 and LSD1. We have noticed that ChIP recovery (% of input) drops slightly on chromatin shared on Pico but at the same time the fold enrichment (ratio pos/neg loci) is usually higher compared to chromatin sheared on old Bioruptors.
Comparing to an old Biorupotrs, Bioruptor Pico is a way more performant. Consequently, a sonication protocol should be re-adapted to Pico by decreasing a sonication time. Usually, 5-10 cycles are already enough for an efficient shearing with fragments in the range 100-300 bp (an ideal range for ChIP-seq) . In some cells type, even shorter sonication time (2-3 cycles) resulted in a nice shearing. It is important to perform a pilot experiment before ChIP to figure out how long to sonicate. The golden rule is always to choose the shortest sonication. For some antibodies, it is worth trying a longer fixation (15-20 min). Regarding ChIP-qPCR, primers sets should be revised and primers amplifying short PCR products (80-100 bp) should be preferred. Longer PCR products could lead to reduced sensitivity in qPCR.

Diagenode-Bioruptor Pico

Thank you for the reply. For some factors, we observed exactly what you said. However, we just cannot chip some other factors. Recently, I run my time-course sonication samples on a western blot together with unsonicated lysate. It turns out the sonication process destroy the epitope of the protein of our interest. You can see the loss of signal as early as 5 min. As a control, we use normal eppendorf tubes which are not very efficient in Bioruptor Pico, it turns out to be okay.

I guess we need to find a fine balance between efficient sonication and not losing protein.

Cheers