6 replies to this topic

[yanks1ny]

To say that we've been having issues with sonication is an understatement. In hopes of getting good chromatin shearing (<1500 bp), we initially decided to use a BioRuptor (that's located in the cold room) [see below for more details]. The first time that we used it (using normal eppendorf tubes) and filled the water bath with a fair amount of ice as per the protocol, it seemed to work well (we saw good fragmentation on a 1% agarose gel). However, the subsequent times, this was no longer the case. First, we tried to eliminate much of the ice. We only added ice in the beginning prior to sonication, stirred it around until it melted to make sure that the water was cold and then just added a miniscule bit of ice to the remaining water. Unfortunately, this did not work, we still saw smearing after running the samples on a gel. Next, we decided to buy the thin walled tubes that they suggested. During the practice run (using untreated cross-linked cells) with 1% SDS buffer, it worked amazingly. After 7.5 minutes, we were getting great shearing as we saw <500 bp fragments. In addition, we started to try to make sure that once we added the SDS buffer to the cells to not place the tubes on ice to limit the chance of SDS coming out of solution. However, when we tried it again using samples for an actual ChIP (sh treated cross-linked cells), we no longer saw this wonderful shearing, but instead, saw a much larger smearing. Finally, we decided to use another lab's Covaris E210 machine. The first time that we used it (again, using untreated cross-linked cells), it worked amazingly well (similar to the 1st time that we used the new tubes on the Bioruptor; after 5 minutes, we saw <500 bp fragments). However, the next time that we used it with samples for an actual ChIP (sh treated cross-linked cells), we no longer saw this effect, but again, saw more of a smear. The only difference that we can think of is a slightly different water level (~20%), but the tubes were still completely submerged in the Covaris machine.


For all the different types of sonication, we've tried using different buffers--0.1% SDS, 1% SDS, and a buffer without SDS (with sodium deoxycholate and N-lauroyl sarcosine)

We are using HeLa S3 cells. For cross-linking: 1% formaldehyde for 10 mins @ RT, then add glycine for 5 min @ RT (at which point we wash in PBS and harvest the cells, generally getting a pellet around 100-140 mg)

We've tried varying the amounts of cells used as well as extracting nuclei first. We've tried using 1/3 of a 15 cm plate (i.e. add buffer to the pellet then divide it 3 ways) or a whole plate. For the Bioruptor, we've been sonicating in ~200 µL. We always use balancing tubes (since there are 6 slots and we normally don't have 6 tubes), which have 200 µL of water in them.

We simply cannot understand the reproducibility issue. Although other people in different lab have had success with both the machines, we know that we can't be going crazy because another person in our lab is having the exact same problem.

Any ideas at all would be extremely helpful...


TL;DR We have had successful sonication using both the BioRuptor and Covaris machines, but we are unable reproduce the good shearing and we are unsure what variables we are changing that could lead to such inconsistency.

[chabraha]

Instead of variability in your shearing, there is the possibility that the variation is coming from the efficency of the reverse cross-linking step............post your protocol? Otherwise I would try (several times on separate days) reproducibly shearing your untreated cells..................and another thought..........I ahve noticed that when I load up too much sheared chromatin onto my agarose gel it affects the migration pattern.............

[yanks1ny]

To reverse cross-link, we add our buffer (1:10 SDS and 1:10 NaHCO3 in water) to our samples (1:10, sample:buffer) then keep the tubes at 65ºC for 12 hours, after which we PCR purify them and run them on a gel.

On a different note, does anyone know if simply treating cells with shRNA can affect chromatin state since we seem to be having different results when we have untreated cross-linked HeLa vs. shRNA-treated cross-linked HeLa?

[chabraha]

hmmmmmm reverse cross-link seems fine............maybe phenol chloroform extract and then precipitate?..................per the shRNA inquiry, I would imagin it depends on the target. There are instances where you can achieve "global chromatin decondensation"..........maybe try transfecting in a control shRNA plasmid and see what happens to your shearing?

Edited by chabraha, Yesterday, 11:56 AM.

[memari]

yanks1ny, on 24 May 2012 - 07:34 AM, said:

To say that we've been having issues with sonication is an understatement. In hopes of getting good chromatin shearing (<1500 bp), we initially decided to use a BioRuptor (that's located in the cold room) [see below for more details]. The first time that we used it (using normal eppendorf tubes) and filled the water bath with a fair amount of ice as per the protocol

Do not you think that you should use TPX tubes (Polymethylpentene (PMP) tubes) instead of normal eppendorf tubes when you use BioRuptor.

https://docs.google.com/file/d/0B15muZJPLjJgbm81UG11X2pUbGlQb3llU2lzampldw/edit?pli=1
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Babak Memari

Edited by memari, Yesterday, 04:08 PM.

[yanks1ny]

Memari: you're right, originally we were using normal eppendorf tubes, but then we switched over to the 1.5 mL TPX tubes. As I had mentioned, during the test run with the untreated cross-linked cells, the sonication looked beautiful with those tubes. However, once we tried again with our shRNA treated cross-linked cells, we no longer saw good sonication.

[yanks1ny]

chabraha: in our actual experiment we have both a shLuc control or an shRNA against our gene of interest. Unfortunately we no longer see good sonication with either samples.