truChIP Chromatin Shearing Kit with Formaldehyde

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Paper describes steps optimized from the manufacturer’s instruction for usage of microvessels.

Protocol tips
Paper describes steps optimized from the manufacturer’s instruction for usage of microvessels.

Publication protocol

Note: We used a commercially available kit, truChIP™ Chromatin Shearing Reagent Kit from Covaris. Here we describe only steps optimized from the manufacturer’s instruction for usage of microvessels.

Add 8 ml of 1 % methanol free PFA solution provided from the kit into a 35mm petridish.

!CAUTION Paraformaldehyde is classified as a known cancer hazard. It should be handled only in a working chemical fume hood with personal protective equipment (PPE) such as lab coats, gloves and face shields.

Transfer the cell strainer containing washed microvessels from step 12 to petridish and incubate for 5 mins at room temperature with gentle shaking.

♦ CRITICAL STEP Make sure the strainer submerged completely into 1% methanol free PFA solution during the crosslinking.

Add quenching buffer provided from the kit as described in manufacturer’s instruction to stop crosslinking.

After a brief wash with 2ml of PBS, retrieve fixed microvessels from the strainer with 10 ml of 1% BSA/PBS, and centrifuge at 2,000 g for 10 mins.

Discard supernatant and resuspend the pellet in the lysis buffer B provided from the kit. We combine microvessels from two brains in 300 μl of lysis buffer B.

￭ Pause Point Microvessels pellet can be stored at −80 °C before processing up to 6 month.

Incubate lysate for 15 mins, 4 °C on a rotator and transfer lysate to the microTUBEs.

Set the CovarisM220 at 5 % duty factor, 50 P.I.P, 200 cpb, and sonicate lysates for 3 mins.

♦ CRITICAL STEP We incorporated a mild sonication steps due to difficulties of pulverizing microvessels. To maximize yield of chromatin extraction, it is critical to disrupt and lyse microvessels properly and completely. After step vii, the structure of microvessels should not be visible.

Centrifuge lysate from step vii at 1,700 g for 5 mins, 4 °C.

Add 600 μl of wash buffer provided from the kit and incubate for 5 mins on a rotator, 4 °C, followed by centrifugation at 1,700 g for 5 mins to collect nuclear extract.

Resuspend pellet in 550 μl of shearing buffer that provided from the kit, and vortex until it is mixed.

Transfer chromatin to the microTUBE and sonicate with the default setting of Low Cell Chromatin Shearing Protocol for M-Series as described in manufacturer’s instruction for 4 or 8 mins.

Process sheared chromatin according to manufacturer’s instruction to check the size of sheared chromatin by electrophoresis using 2 % agarose gel or by BioAnalyzer.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Mouse - Brain using truChIP Chromatin Shearing Kit with Formaldehyde from Covaris.

Manufacturer protocol

Download the product protocol from Covaris for truChIP Chromatin Shearing Kit with Formaldehyde below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Mouse - Brain using truChIP Chromatin Shearing Kit with Formaldehyde from Covaris. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.