CHiP on chip Drosophila:Preparation of fixed chromatin from whole Drosophila

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WARNING: This protocol is in development --Johncumbers 10:56, 18 April 2006 (EDT)

Contents

Overview

The aim of the method is to isolate fixed chromatin from Drosophila whole flies. Essentially, the chromatin is fixed, the nuclei are then purified and lysed before the chromatin is fragmented by sonication. This protocol was developed by Rob White's laboratory and is here adapted for whole flies.

to add: fly collection details, time collected weight of flies for each chromatin prep.

Equipment and reagents

  • Sigma 4K10 bench top centrifuge
  • Standard microfuge tube bench top centrifuge
  • Formaldehyde (40% solution) 'Analar grade' (BDH; Cat. No. 101134A)
  • 50 ml Falcon tubes
  • 13.5 ml screw−capped conical base tubes (Bibby Sterlin; Cat. No. 144AS)
  • 212−300 micron glass beads (Sigma; Cat No. G−1277)
  • Heat Systems Ultrasonic Liquid Processor XL sonicator
  • Wheaton Dounce homogeniser

Solutions and reagents

10 x PBS, pH 7.4:

  • 80 g Sodium chloride
  • 2 g Potassium chloride
  • 14.4 g Disodium hydrogen orthophosphate
  • 2.4 g Sodium dihydrogen orthophosphate
    • Make to a total volume of 1 litre.

Crosslinking solution:

  • 50 mM HEPES, pH 8.0
  • 1 mM EDTA.Na2 ·
  • 0.5 mM EGTA
  • 100 mM NaCl

PBS/Triton:

  • 1 x PBS containing 0.01% Triton X−100
  • Add 0.5 ml of 1% Triton stock to 50 ml 1 x PBS
    • (+ or − protease inhibitors)
  • PBS/Glycine/Triton:
  • 1 x PBS/Triton containing 125 mM glycine.

Cell Lysis Buffer:

  • 5 mM PIPES, pH 8.0
  • 85 mM Potassium chloride
  • 0.5% Nonidet P40 (NP40)
  • + protease inhibitors

Autoclave without NP40 and then add appropriate amount of NP40 from 10% stock

Nuclear Lysis Buffer:

  • 50 mM Tris.HCl, pH 8.1
  • 10 mM EDTA.Na2
  • 1% SDS (this seems too hight, it might froth up when sonicating)
  • + protease inhibitors

Autoclave without SDS and then add appropriate amount of SDS from 10% stock.

Notes

  1. unsure how may flies are needed
  2. unsure if it will work in whole flies, should do
  3. unsure of quantities at the moment

Preparation of fixed chromatin from whole Drosophila

  1. Use dounce homogenizer to homogenize flies
  2. Transfer -weight-g of flies to a Falcon tube and add -X-ml PBS/Triton to wash the embryos.
  3. Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos.
  4. Discard supernatant and add 10 ml CHiP on chip Drosophila:cross−linking solution, 487 μl 40% formaldehyde and 30 ml n−heptane (Heptane not needed for whole flies, used to get through coreon(?).) Shake vigorously (by hand works well...but tiring) at room temperature for 15 minutes.
  5. Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet flies and discard supernatant.
  6. Resuspend in 30 ml PBS/Glycine/Triton, best to resuspend in 3 ml of buffer and then add the rest. Then allow the flies to sediment.
  7. Remove supernatant and add 50 ml ice−cold PBS/Triton and resuspend. Best to add a small volume of the buffer to resuspend the flies. Allow the flies to sediment.
  8. Remove supernatant and resuspend in 15 ml ice−cold PBS/Triton + protease inhibitors, again resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle B.
  9. Centrifuge at 400 x g (1500 rpm) for 1 minute and transfer supernatant to a fresh tube. Centrifuge at 1100 x g (2495 rpm) for 10 minutes at 4 °C and discard supernatant. Resuspend in 15ml ice−cold Cell Lysis Buffer with protease inhibitors, best to resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle A. Transfer 2 equal aliquots into 13.5 ml screw−capped conical base tubes.
  10. Centrifuge at 2000 x g (3365 rpm) for 4 minutes at 4 °C to pellet the nuclei. Resuspend in 1 ml of ice−cold Drosophila CHiP on chip:Nuclear Lysis Buffer with protease inhibitors, incubate for 20 minutes at 4 °C.
  11. Add 2 ml ice−cold Nuclear Lysis Buffer with protease inhibitors and 0.3 g acid washed 212−300 micron glass beads.
  12. Sonicate on ice following the regime below to produce chromatin fragments with an average size of 500 bp using a sonicator fitted with a microtip. This step should be calibrated for individual sonicators to generate chromatin of an appropriate size.
    • 1 x 30 seconds on level 3
    • 5 x 30 seconds on level 4
    • Between each sonication rest on ice for 90 seconds
  13. Transfer to microfuge tubes and centrifuge at 16,000 x g (13000 rpm) in a microfuge for 10 minutes at 4 °C.
  14. Take the supernatant (fixed sheared chromatin) and transfer to cryotubes, e.g., 200 μl aliquots, and flash freeze in liquid nitrogen. Store at −80 °C.
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