Overnight cultures were grown in LB 37 °C/225rpm and then subcultured 1:100. Subcultures were grown in LB 37 °C/225rpm to an OD600 of 0.5-0.7. Initial RNA purification: RNA was purified from cells using a modified version of the hot phenol method that has been described previously. 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°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.   DNase treatment and Ribosomal RNA removal: A 50uL reaction including 8-10ug RNA, 5uL of 10X buffer and 1uL DNase enzyme (Ambion) was incubated 30 min at 37 °C.  The reaction was stopped by adding 352uL DEPC H2O + 134uL acid phenol:chloroform + 268uL chloroform:isoamyl alcohol 24:1.  After spinning in a microcentrifuge at max speed for 6 minutes, the upper phase was transferred to new microcentrifuge tube containing 40uL 3M Na Acetate + 2uL glycogen (20mg/mL stock). 1mL of ice-cold EtOH was added and the RNA was precipitated at -20 °C overnight or 15min at -80 °C.  The samples were microcentrifuged for 30 min at 4 °C at max speed and then washed twice with 0.7mL of 70% EtOH, microcentrifuging 5 min at max speed at 4 °C between each wash. The pellets were dried at room temperature and resuspended in 15uL nuclease free water.  The ribosomal RNA was removed using the RiboZero kit from EpiCentre.  The magnetic beads were washed 2x in RNase free water, and then resuspend in Resuspension Solution.  32.5uL beads and 0.5uL RNase inhibitor were added to each tube. In final volume of 20uL, the following mixture was prepared: 2.5ug DNase treated RNA + 2uL Ribozero reaction buffer + 4uL Ribozero rRNA removal solution.  The mixture was incubated at 68 °C for 10 min followed by 20C for 5 min.  The RNA was then added to the prepared beads and mixed by pipetting and vortexing for 10 s.  The samples were incubated at RT for 5 min.  The samples were then vortexed for 5 s then incubated at 50 °C for 5 min.  The tubes containing magnetic bead/RNA mixture were placed on a magnetic stand for 1 min. The RNA (depleted of ribosomal RNA) was transferred to a new tube.  The volume was brought up to 180uL with DEPC H2O.  18uL 3M Na Acetate + 2uL glycogen (20mg/mL stock) were added to each RNA sample.  0.6mL ice cold EtOH was added to each sample and the RNA was precipitated at -20 °C overnight.  The samples were microcentrifuged for 30 min at 4 °C at max speed and then washed twice with 0.7mL of 70% EtOH, microcentrifuging  5 min at max speed at 4 °C between each wash. The pellets were dried at room temperature and resuspended in 8uL nuclease free water. Prepare libraries using EpiCentre ScriptSeq v2 kit following kit protocol.  Fragment RNA and anneal cDNA synthesis primer in 12uL reaction containing nuclease free water + 40ng rRNA depleted RNA + 1uL RNA Fragmentation solution + 2uL cDNA Synthesis primer.  Incubate at 85 °C for 5 min then place tubes on ice.  Synthesize DNA by adding 3uL cDNA Synthesis Premix + 0.5uL 100mM DTT + 0.5uL StarScript Reverse Transcriptase.  Incubate at 25 °C for 5 min then 42 °C for 20 min.  Cool tubes to 37 °C.  Add 1uL Finishing solution and incubate at 37 °C for 10 min then 95 °C for 3 min.  Cool tubes to 25 °C then add 7.5uL Terminal Tagging Premix + 0.5uL 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 0.75mL 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 23.4uL Buffer EB to column, incubate 1 min at room temp, then spin 1 min at max speed.  Amplify library by setting up 50uL PCR using 22.5uL purified DNA.  Add 25uL FailSafe PCR Premix E + 1uL Forward PCR primer + 1uL ScriptSeq Index PCR Primer + 0.5uL 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 50uL 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 0.2mL of 80% EtOH, let stand 1 min then remove supernatant.  Air dry beads at room temp for 15 min.  Add 12uL 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: genotype
no deletions
Experimental Factor: replicate
2