alias: E-MTAB-3699:pBAD24 Rep1
title: Illumina HiSeq 2000 sequencing; Genome-Scale Mapping of Escherichia coli Ď54 Reveals Widespread, Conserved Intragenic Binding
design: Genome-Scale Mapping of Escherichia coli Ď54 Reveals Widespread, Conserved Intragenic Binding
library_construction_protocol: Overnight cultures were started from single colonies in 2 mL M9+0.4% glycerol at 30 degrees Celsius shaking at 225 rpm and grown for ~36 hours.  40 ml subcultures were grown in M9+0.4% glycerol at 30 degrees Celsius to an OD600 of approximately 0.5. Arabinose was added to the culture at a final concentration of 0.2% and incubated for 10 minutes at 30 degrees Celsius and shaking at 225 rpm. Hot phenol RNA prep:  transfer 1mL of subculture to two microcentrifuge tubes.  Spin each 1mL at max speed for 1 min and remove supernatant.  Resuspend each in 0.4mL ethanol+5% acid phenol/chloroform, spin 1 min at max speed and remove supernatant.  Resuspend each in 1mL 1x TBS, spin 1 min at max speed and remove supernatant.  Resuspend each in 0.25mL RNA Lysis Buffer (2% SDS, 4mM EDTA), combine and boil samples for 3 min.  Add 0.5mL 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 (0.4mL) chloroform:isoamyl alcohol 24:1 and spin 6 min at max speed.  Transfer upper phase to new phase lock tube, add equal volume (0.4mL) chloroform:isoamyl alcohol 24:1 and spin 6 min at max speed.  Transfer upper phase to new tube containing 40uL 3M Na Acetate+2uL glycogen (20mg/mL stock).  Add 1mL 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 0.7mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 °C and remove supernatant.  Wash again with 0.7mL 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 15uL nuclease free water.  DNase treat RNA - set up 50uL reaction including 8-10ug RNA, 5uL of 10X buffer and 1uL DNase enzyme (Ambion).  Incubate 30 min at 37 °C.  Stop reaction by adding 352uL DEPC H2O + 134uL acid phenol:chloroform + 268uL 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 40uL 3M Na Acetate + 2uL glycogen (20mg/mL stock).  Add 1mL 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 0.7mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 °C and remove supernatant.  Wash again with 0.7mL 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 15uL 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 32.5uL beads to each tube then add 0.5uL RNase inhibitor.  Mix together in final volume of 20uL:  2.5ug DNase treated RNA + 2uL Ribozero reaction buffer + 4uL 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 180uL with DEPC H2O, then add 18uL 3M Na Acetate + 2uL glycogen (20mg/mL stock).   Add 0.6mL 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 0.7mL of 70% EtOH (in DEPC H2O), spin 5 min at max speed at 4 °C and remove supernatant.  Wash again with 0.7mL 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 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.
identifiers.namespace: WADSWORTH CENTER, NY DEPARTMENT OF HEALTH
identifiers.id: E-MTAB-3699:pBAD24 Rep1
identifiers.namespace: Wadsworth Center, NY Department of Health
identifiers.id: E-MTAB-3699:pBAD24 Rep1
attributes.tag: Experimental Factor: pBAD24
attributes.value: genotype