162
edits
Changes
no edit summary
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 (60% amplitude--this 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.
