Berglund:Footprinting

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