Illumina HiSeq 2000 sequencing; SuhB associates with Nus factors to facilitate 30S ribosome biogenesis in Escherichia coli
SuhB associates with Nus factors to facilitate 30S ribosome biogenesis in Escherichia coli
Overnight cultures were started from single colonies in 2 mL LB at 37 C, shaking at 225rpm.  Subcultures were set up at 1:100 in 20 mL LB and grown at 37 C, shaking at 225rpm until OD600 reached 0. Hot phenol RNA prep:  transfer 1 mL of subculture to microcentrifuge tube.  Spin 1 mL at max speed for 1 min and remove supernatant.  Resuspend in 0.4 mL ethanol+5% acid phenol/chloroform, spin 1 min at max speed and remove supernatant.  Resuspend in 1 mL 1x TBS, spin 1 min at max speed and remove supernatant.  Resuspend in 0.5 mL RNA Lysis Buffer (2% SDS, 4 mM EDTA) and boil samples for 3 min.  Add 0.5 mL acid phenol/chloroform to each tube and incubate for 6 min at 65 C.  Incubate 5 min on ice then spin tubes at max speed for 6 min.  Transfer upper phase to phase lock tube, add equal volume (0.4 mL) chloroform:isoamyl alcohol 24:1 and spin 6 min at max speed.  Transfer upper phase to new phase lock tube, add equal volume (0.4 mL) chloroform:isoamyl alcohol 24:1 and spin 6 min at max speed.  Transfer upper phase to new tube containing 40 uL 3M Na Acetate+2 uL glycogen (20mg/mL stock).  Add 1 mL ice cold EtOH and precipitate at -20 C overnight or 15 min at -80 C.  Spin samples for 30 min at 4 C at max speed.  Pipet off supernatant.  Wash with 0.7 mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 C and remove supernatant.  Wash again with 0.7 mL of 70% EtOH (in DEPC H2O), spin for 5 min at max speed at 4 C and pipet off supernatant.  Dry pellet at room temperature and resuspend in 15 uL nuclease free water.  DNase treat RNA - set up 50 uL reaction including 8 ug RNA, 5 uL of 10X buffer and 1 uL DNase enzyme (Ambion).  Incubate 30 min at 37 C.  Stop reaction by adding 352 uL DEPC H2O + 134 uL acid phenol:chloroform + 268 uL chloroform:isoamyl alcohol 24:1.  Transfer all to phase lock tube and spin at max speed for 6 min.  Transfer upper phase to new microcentrifuge tube containing 40 uL 3M Na Acetate + 2 uL glycogen (20mg/mL stock).  Add 1 mL ice cold EtOH and precipitate at -20 C overnight or 15 min at -80 C.  Spin samples for 30 min at 4 C at max speed.  Pipet off supernatant.  Wash with 0.7 mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 C and remove supernatant.  Wash again with 0.7 mL of 70% EtOH (in DEPC H2O), spin for 5 min at max speed at 4 C and pipet off supernatant.  Dry pellet at room temperature and resuspend in 15 uL nuclease free water.  Remove ribosomal RNA using the RiboZero kit from EpiCentre - wash magnetic beads 2x in RNase free water, then resuspend in Resuspension Solution.  Add 32.5 uL beads to each tube then add 0.5 uL RNase inhibitor.  Mix together in final volume of 20 uL:  2.5 ug DNase treated RNA + 2 uL Ribozero reaction buffer + 4 uL Ribozero rRNA removal solution.  Incubate at 68 C for 10 min followed by RT for 5 min.  Add RNA to beads, mix by pipetting and vortexing for 10 s.  Incubate at RT for 5 min.  Vortex for 5 s then incubate at 50 C for 5 min.  Place tubes on magnetic stand for 1 min and transfer RNA to new tube.  Bring volume to 180 uL with DEPC H2O, then add 18 uL 3M Na Acetate + 2 uL glycogen (20mg/mL stock).   Add 0.6 mL ice cold EtOH and precipitate at -20 C overnight.  Spin samples for 30 min at 4 C at max speed.  Pipet off supernatant.  Wash with 0.7 mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 C and remove supernatant.  Wash again with 0.7 mL of 70% EtOH (in DEPC H2O), spin for 5 min at max speed at 4 C and pipet off supernatant.  Dry pellet at room temperature and resuspend in 8 uL nuclease free water. Prepare libraries using EpiCentre ScriptSeq v2 kit following kit protocol.  Fragment RNA and anneal cDNA synthesis primer in 12 uL reaction containing nuclease free water + 40 ng rRNA depleted RNA + 1 uL RNA Fragmentation solution + 2 uL cDNA Synthesis primer.  Incubate at 85 C for 5 min then place tubes on ice.  Synthesize DNA by adding 3 uL cDNA Synthesis Premix + 0.5 uL 100mM DTT + 0.5 uL StarScript Reverse Transcriptase.  Incubate at 25 C for 5 min then 42 C for 20 min.  Cool tubes to 37 C.  Add 1 uL Finishing solution and incubate at 37 C for 10 min then 95 C for 3 min.  Cool tubes to 25 C then add 7.5 uL Terminal Tagging Premix + 0.5 uL DNA Polymerase to terminally tag the cDNA.  Incubate at 25 C for 15 min, 95 C for 3 min, then cool to 4 C.  Purify the cDNA using the Qiagen MinElute PCR Purification Kit - Add 5V Buffer PB to sample and transfer all to MinElute column.  Spin 1 min at max speed.  Discard flow through.  Add 0.75 mL Buffer PE to column and spin 1 min at max speed.  Discard flow through.  Spin column for 1 min at max speed then transfer column to new tube.  Add 23.4 uL Buffer EB to column, incubate 1 min at room temp, then spin 1 min at max speed.  Amplify library by setting up 50 uL PCR using 22.5 uL purified DNA.  Add 25 uL FailSafe PCR Premix E + 1 uL Forward PCR primer + 1 uL ScriptSeq Index PCR Primer + 0.5 uL FailSafe PCR Enzyme (1.25U).   Heat at 95 C for 1 min, then 12 cycles of 95 C for 30 sec, 55 C for 30 sec and 68 C for 3 min.  Incubate at 68 C for 7 minutes.  Purify sample using AMPure XP Purification protocol.  Add 50 uL beads to each pcr sample.  Mix by pipetting 10x.  Transfer beads+sample to new tube and incubate at room temp for 15 min.  Put tubes on magnetic stand for 5 min then remove supernatant.  Wash beads 2x, each time with 0.2 mL of 80% EtOH, let stand 1 min then remove supernatant.  Air dry beads at room temp for 15 min.  Add 12 uL nuclease free water, remove tubes from magnetic stand and resuspend beads in water.  Incubate at room temp for 2 min.  Place tubes on magnetic stand for 5 min.  Library is in supernatant.  Transfer to new tube.  Quantify using Qubit.
Experimental Factor: thyA nusB::thyA
genotype
Experimental Factor: deletion nusB
genetic modification