Hfq-Flag_Log_Phase_CL_Repeat2
Protocols: E .coli Strains MG1655 and HM34 were grown overnight in LB at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB, and re-grown with shaking at 37 °C to log phase (OD600 = 0.5) cells were exposed to 800 mJ of 254 nm UV irradiation (Stratalinker® UV Crosslinker 1800) on a metal block cooled to -20 oC To trim the exposed parts of the RNAs, beads were resuspended in 200 μl of lysis buffer supplied with 0.5 unit of RNase A/T1 mix (Thermo Scientific) without RNase inhibitor, and incubated for 3-15 min at 22 °C or 37 °C. RNase digestion buffer was removed and the beads were washed three times with 200 μl lysis buffer supplied with 0.1 U of SUPERase INTM RNase Inhibitor (Life technologies) at 4 oC. 5'OH ends were phosphorylated and 2'P / 3'P ends were dephopsphorylated by incubation with 40 units of T4 PNK (New England Biolabs) in 80 μl of PNK buffer for 2 hr at 22 °C. PNK reaction mixture was removed and the beads were washed three times with 200 μl lysis buffer at 4 oC. Neighbouring RNAs were ligated overnight using 216 units of T4 RNA Ligase 1 (New England Biolabs) in 80 μl ligase buffer. Ligation mixture was removed and the beads were washed three times with 200 μl lysis buffer at 4 oC. Ligation of 3' barcoded adaptors. First cDNA strand was generated using SuperScript III (Life Technologies) RNA was extracted according to the standard LS-TriReagent protocol (Sigma) RNAtag-Seq protocol (Shishkin et al., 2015)
ENA first public
2016-07-30
ENA last update
2015-09-22
External Id
SAMEA3554120
INSDC center alias
The Hebrew University of Jerusalem, Israel
INSDC center name
The Hebrew University of Jerusalem, Israel
INSDC first public
2016-07-30T17:01:21Z
INSDC last update
2015-09-22T15:49:33Z
INSDC status
public
Submitter Id
E-MTAB-3910:Hfq-Flag_Log_Phase_CL_Repeat2
broker name
ArrayExpress
experimental process
cross link RNA
genotype
Hfq-flag
growth condition
log phase
sample name
E-MTAB-3910:Hfq-Flag_Log_Phase_CL_Repeat2
strain
K-12
substrain
MG1655