162
edits
Changes
→Sonication of Tissue
The tissue fixation portion of this protocol was taken from the Ren Lab ENCODE protocol for brown adipose tissue and can be found here [https://www.encodeproject.org/documents/3125496b-c833-4414-bf5f-84dd633eb30d/@@download/attachment/Ren_Tissue_Fixation_and_Sonication_v060614.pdf].
=Before You Start=
==Buffers and Solutions Needed==
Mass of tissue needed: TBD
Crosslinking Buffer
Lysis Buffer
TE
2.5 M Glycine
Cold 1x PBS
ChIP Elution Buffer (make fresh)
LiCl Wash Buffer
==Pulverization of Tissue==
# Place the sample back on dry ice.
#Clean the mortar and pestle with 10% bleach and 70% ethanol between samples
''You can continue or store at -80C.''
==Cross-linking of Tissue==
# Transfer tissue into a 15mL conical tube using a clean spatula or pipette
# Add cold 1x PBS up to 5mL to all tubes as you go.
# Add 0.5mL cross linking buffer and rotate the tube at room temperature for 20 min. (Add this to all samples at the same time)
# Stop the crosslinking reaction by adding 0.275mL of 2.5 M glycine to a final concentration of 0.125 M.
# Rotate at room temperature for 5 min.
# Centrifuge samples at low speed (15 min at 2000 x g/RCF).
# Decant the supernatant and was once with 5mL cold 1x PBS.
# Centrifuge at low speed (10 min at 2500 x g/RCF).
# Decant the supernatant.
# Store cells at -80C or proceed to sonication.
==Sonication of Tissue==# Resuspend fixed cells in 50-150uL lysis buffer(depending on amount of tissue) and incubate for 10 min on ice.# Dilute to 500-1.5mL cold 1x TE (this is based on the amount of lysis buffer you used).# Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each (this is around 60% amplitude--but it ranges depending on how much tissue so play around with amplitude for your sample to get near 5W), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.*If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each. # Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant and nano drop samples and calculate the amount needed for 25ug of chromatin. # Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below.
===Immunoprecipitation===