title: Illumina HiSeq 2000 sequencing; TSSdelta rppH vsdelta rppHdelta hns double mutant
design: TSSdelta rppH vsdelta rppHdelta hns double mutant
library_construction_protocol: Overnight culures were grown in LB medium  at 30C. Subculture the overnight in 1:100 diution in LB at  30C. Grow cells to mid log OD and harvest RNA. RNA was purified from cells using a modified version of the hot phenol method. 1 ml cells were mixed with 400 ul ice-cold 95% ethanol and 5% phenol/chloroform/isoamyl alcohol (25:24:21 mix). Cells were pelleted in a microcentrifuge for 1 minute at full speed and washed once with TBS. Cell pellets were resuspended in 400 ul RNA lysis buffer (2% SDS, 4 mM EDTA) and boiled for 3 minutes. 400 ul acid phenol/choroform/isoamyl alcohol mix (pH 4.3) was added and incubated at 65 degrees C for 6 minutes and on ice for 5 minutes. Samples were centrifuged and the aqueous layer was extracted once more with phenol/choroform/isoamyl alcohol mix (pH 4.3). RNA was precipitated with 1 ml 100% ethanol and 40 ul 3 M sodium acetate. RNA was pelleted in a microcentrifuge for 10 minutes at full speed and washed once with room-temperature 75% ethanol. RNA pellets were air-dried and resuspended in water. A 50 uL reaction including 8-10 ug RNA, 5 uL of 10X buffer and 1 uL DNase enzyme (Ambion) was incubated 30 min at 37C.  The reaction was stopped by adding 352 uL DEPC H2O + 134 uL acid phenol:chloroform + 268 uL chloroform:isoamyl alcohol 24:1.  After spinning in a microcentrifuge at max spped for 6 minutes, the upper phase was transferred to new microcentrifuge tube containing 40 uL 3 M Na Acetate + 2uL glycogen (20 mg/mL stock). 1 mL of ice-cold EtOH was added and the RNA was precipitated at -20C overnight or 15 min at -80 C.  The samples were microcentrifuged for 30 min at 4C at max speed and then washed twice with 0.7 mL of 70% EtOH, microcentrifuging 5 min at max speed at 4C between each wash. The pellets were dried at room temperature and resuspended in 15 uL nuclease free water.  Ribosomal RNA was removed using the RiboZero kit for gram negative bacteria (Epicentre) according to the manufacturer's instructions. TSS libraries were prepared using an established protocol (Singh and Wade, Methods in Molecular Biology, in press). Two independent biological replicates were performed for each strain.
attributes.tag: Experimental Factor: genotype
attributes.value: delta rppH