Berglund:Footprinting

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Transcribe RNA using SHAPE protocol: http://openwetware.org/wiki/Berglund:cold_SHAPE_method(Amy)
Clean using the Q column: http://openwetware.org/wiki/Berglund:RNA_column_purification

remove the 5' phosphate
SAP the RNA:

10X kinase buffer 1ul
6µg of RNA
water
SAP 1ul
10µl final volume

put the hot phosphate onto the RNA
Kinase the RNA:

SAP mix 10ul
10x kinase buffer 1ul
water 2ul
hot ATP 5ul
T4 polynucleotide kinase 2ul
20µl final volume

Heat at 37° for 30 minutes
Add 20µl of denaturing formamide buffer
heat denature at 95° for 2 minutes

Load on a 6% denaturing gel and run for ~ 1 hr.

Take apart plates
put saran wrap on top of gel
put phospho imager plate on for 3 minutes
scan on the STORM phosphoimager in the gel room (take a thumb drive with you
Print off picture
Cut out band of RNA

Elute RNA out of gel band for 15 minutes in 1ml 300mM NaOAc pH5.2
Take supernate off band and put into 1.5ml eppendorf tube
Put 1ml more of 200mM NaOAc pH 5.2 onto the gel slice and let elute for 45 minutes
Add 1µl of glycogen to supernate (carrier to help RNA come down)
Add 2.5ml of Ethanol and spin down hard
Repeat on the second ml of eluted RNA

          • Do all spins into the same tube so that you get one pellet

Resuspend RNA in 10µl of low TE buffer
Immediately start probing with Rnase/ alkaline hydrolysis

Choose your Rnase that you want (single verus double stranded)
Follow manufacture's instructions
Run final products on 15% denaturing gel(http://openwetware.org/wiki/Berglund:Acrylamide) sequencing length (2.5 feet)with 0.5mM spacers and 1.5cm comb sized teeth
Run for 3 hours at 50W
Put on phosphor plates overnight and then scan

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