Ambion Turbo DNase (Thermofisher – Cat#AM2239)
Recombinant RNasin RNase inhibitor (Promega – cat# N2515)
Ultrapure DNase/RNase free dH2O (Thermofisher Cat# 10-977-015)
Phenol/chloroform with acidic pH (we use Fisher cat#BP1752I-100 without adding the additional buffer, but other should work)
3 M sodium acetate (Thermofisher – Cat# AM9740)
Glycogen (Thermofisher – Cat# AM9510)
100% Ethanol (we use VWR Cat# 89125-170, but others should be fine)
T4 RNA ligase (Ambion Cat#AM2141)
Invitrogen M-MLV reverse transcriptase (Thermofisher – Cat# 28-025-013)
Random decamers (Thermofisher – Cat#AM5722G)
dNTPs (we use Thermofisher – Cat#FERR0181, but others should work; keep RNase free) – make a working stock of 25 mM
Taq DNA polymerase with standard Taq buffer (New England Biolabs – Cat#M0273)
RACE RNA adapter – make working stocks of 0.3 µg/µl
GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA
Primers – make working stocks of 10 µM:
5’ RACE outer forward primer (binds RNA adapter):
5’ RACE inner forward primer (binds RNA adapter sequences and allows for Gibson cloning of fragments into the EcoRI site of pCDNA3.1(+)):
GGATCCACTAGTCCAGTGTGGTGGAATTCGAACACTGCGTTTGCTGGCTTTGATG
Gene-specific outer and inner reverse primers – design ~200 nt downstream of expected cleavage site; should allow for nested PCR.
Water bath or heat block set at 37°C
Refrigerated microcentrifuge