title: del-psaE rep2
organism: Yersinia pestis
description: Protocols: Bacteria cultured overnight in pH 8.0 of HIB media at 28 °C were collected and reinoculated in the pH 6 of HIB media and cultured at 37 °C for 4 h. One ml cells were pelleted in a microcentrifuge for 1 min at full speed and washed once with Tris-buffered saline. RNA was purified from bacterial cells using the Quick-RNA™ Miniprep Plus Kit (Zymo Research). DNase treat RNA - set up 50uL reaction including 6 ug RNA, 5 uL of 10X buffer and 1 uL DNase enzyme (Ambion). Incubate 45 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 (20 mg/mL stock). Add 1 mL ice cold EtOH and precipitate at -20 °C overnight or 15min 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 Illumina - wash magnetic beads 2x in RNase free water (same volume as bead volume), then resuspend in Resuspension Solution (30 uL * number of reactions). Add 32.5 uL beads to each tube then add 0.5 uL RNase inhibitor. In new tube, 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 15 uL nuclease free water. Prepare libraries using Illumina 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 14 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-04T04:03:18Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-09-24T14:19:25Z
attributes.tag: External Id
attributes.value: SAMEA5970603
attributes.tag: INSDC center name
attributes.value: Wadsworth Center, NYS Department of Health
attributes.tag: INSDC first public
attributes.value: 2019-10-04T04:03:18Z
attributes.tag: INSDC last update
attributes.value: 2019-09-24T14:19:25Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8369:del-psaE rep2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: delta psaE
attributes.tag: growth condition
attributes.value: HIB media
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-8369:del-psaE rep2
attributes.tag: scientific_name
attributes.value: Yersinia pestis
attributes.tag: strain
attributes.value: KIM6