Escherichia coli str. K-12 substr. MG1655
Protocols: Bacterial samples were collected after 90 min tetracycline treatment. GB1+pJTW100 bacterial strain was subcultured 1/100 in LB media supplemented with 30 ug/ml of chloramphenicol. Cells were grown at 37oC with shaking. GB1+pJTW100 culture was split at OD600~1.2; one set was left untreated and the second set was treated with 0.2 ug/ml of tetracycline for 90 min;After 90 min, 500 μl of each sample (tetracycline treated and untreated) was additionally treated with 13.3 μl of DMSO or 13.3 µL of 250 mM 1M7 (MedChem Express HY-D0913) SHAPE reagent (in DMSO) for 3 min as described in Watters et al, 2016; cells were harvested immediately for subsequent RNA extraction. Adapted from Watters et al, 2016 with minor modifications:- After 3 min DMSO/1M7 treatment, spin the tubes at ≥ 13,000 rpm for 1 min and remove the used media.- Add 250 µL TRIzol reagent to each, resuspend and add another 750 µL of TRIzol reagent. Shake vigorously for ≥ 5 s to mix.- Incubate the TRIzol-containing tubes for ≥ 5 min at room temperature.- Add 200 µL chloroform to each tube, shake vigorously for 10 s, and incubate at room temperature for another 2 min.- Centrifuge the TRIzol-containing tubes at 13,000 rpm and 4°C for 15 min.- Carefully transfer the clear aqueous phases (up to 400 µL) to new 1.7 mL microcentrifuge tubes.- Add 500 µL cold 100% isopropyl alcohol and 2 µL of 20 mg/mL glycogen, mix by inverting the tubes 6-8 times. Incubate at room temperature for 10 min.- Spin the tubes at ≥ 13,000 rpm and 4°C for 15 min. Aspirate the alcohol and wash with 1 mL 70% EtOH (in DEPC H2O) (or store at -80 to -20 oC). Respin the tubes for 2 min and remove the EtOH, allowing traces of remaining EtOH to air dry.- Resuspend each resulting RNA sample in 12 µL RNase-free water. Adapted from Watters et al, 2016 with minor modifications:*Reverse Transcription- Add 3 µL of 50 nM reverse transcription primer for topAI gene (JW9200).- When the thermal cycler block is preheated to 95°C, place each tube on the thermal cycler, and advance the steps: 2 min 95°C, 5 min 65 °C.- During the first 7 min of incubation (2x Denature), mix 6 µL of SSIII Master Mix and 1 µL of 0.5X SSIII reverse transcriptase (diluted in its storage buffer) for each RNA sample in a microcentrifuge tube. Super Script III master mix: Mix 4 volumes of 5X SSIII First Strand Buffer (supplied with SSIII), 1 volume of 0.1 M DTT, and 1 volume of 10 mM dNTPs.- At the end of the first 65°C step (2nd Denature), move all the tubes to ice for 30 s. Then add 7 µL of the SSIII Master Mix/reverse transcriptase mix to each tube, mix well, and return to ice. Once all of the RNAs have received the 7 µL, return all the tubes to the thermal cycler and continue the thermal cycler procedure: 1 min 45 °C, 25 min 52 °C, 5 min 65 °C.- Upon completion of the RT protocol, remove all the tubes to ice and add 1 µL of 10 M NaOH.- Incubate the tubes at 95°C for 5 min to hydrolyze the RNA.- Partially neutralize the solutions by adding 5 µL of 1 M HCl.- Ethanol precipitate by adding 78 µL of ice-cold 100% ethanol to each tube, inverting 6-8 times, and incubating at -80°C for 15 min. Transfer to 1.7 mL Eppendorf tubes.- Centrifuge the tubes at ≥ 13,000 rpm and 4°C for 15 min. Aspirate the ethanol and wash with 1 mL of 70% ethanol. Respin for 2 min, aspirate, respin for 15 min again, and aspirate the remaining ethanol. Allow to air dry.- Resuspend each cDNA in 22.5 µL of RNase-free water.*A_adapter_B Ligation & Purification- Transfer cDNA to PCR tubes. Make a CircLigase mastermix, per 1 reaction: 3 µL of 10x CircLigase reaction buffer, 1.5 µL 50 mM MnCl2, 1.5 µL 1 mM ATP, 0.5 µL of 10 µM A_adapter_b (ssDNA adapter JW9186) and 1 µL of CircLigase to each tube with cDNA.- Incubate at 60°C for 2 hrs, followed by a 10 min incubation at 80°C to inactivate CircLigase I.- After the ligation is complete, transfer reaction to a new 1.7 mL tube, add 70 µL RNase-free water, 10 µL 3.0 M sodium acetate pH 5.5, 1 µL 20 mg/mL glycogen, and 300 µL of ice-cold 100% ethanol. Mix.- Incubate at -80°C for 30 min then spin at ≥ 13,000 rpm at 4°C for 30 min. Aspirate the ethanol, re-spin for 2 min, and aspirate any remaining ethanol. Resuspend each cDNA in 20 µL water.- Resuspend a bottle of Agencout AMPure XP beads. Add 36 µL of bead slurry to each tube containing cDNA and mix well by pipetting up and down.- Let the bead-dsDNA slurry stand at room temperature for 5 min, then magnet for 2 min, remove the supernatant and wash with fresh 70% EtOH twice on the magnetic stand. Dry the beads and resuspend in 20 µL TE buffer to elute, magnet again and keep the supernatant with the cDNA.*dsDNA Library Construction- Mix the following PCR for each sample: 3 µL ssDNA library, 2 µL of 0.1 µM selection primers (JW9178 (+) or JW9179 (-) (topAI)), 0.25 µL of 100 µM Illumina indexing primer; PCR mastermix: 10 µL of 5X Phusion reaction buffer (NEB), 0.2 µL of 25 mM dNTPs, 0.5 µL (1 U) Phusion Polymerase, and 33.8 µL H2O (Total reaction volume = 50 µL). PCR reaction conditions: 1x 98 °C for 30s, 15x 98 °C for 15s followed by 65 °C for 30s and 72 °C for 30s. At the end of the cycle 15, add 0.25 µL of 1 μM PE_forward TruSeq (JW9175) primer for another 15 cycles, ending with a 72 °C 5 min extension. The identical Illumina indexing primers can be used for the same sample that was treated with DMSO or 1M7; negative (-, DMSO) and positive (+, 1M7) channels will be separated bioinformatically based on their handles on selection primers.- Chill the reactions at 4°C for 5 min.- Add 0.25 µL (5 U) of ExoI to each library reaction.- Incubate at 37°C for 30 min.- Resuspend a bottle of Agencout AMPure XP beads. Add 90 µL of bead slurry to each tube containing cDNA and mix well by pipetting up and down.- Continue purification of dsDNA libraries as in Step 31, eluting in 20 µL H2O.- Measure the concentration of each dsDNA library using Qubit.
ENA-FIRST-PUBLIC
2019-10-03T06:24:46Z
ENA-LAST-UPDATE
2019-09-23T16:19:04Z
External Id
SAMEA5795549
INSDC center name
Wadsworth Center
INSDC first public
2019-10-03T06:24:46Z
INSDC last update
2019-09-23T16:19:04Z
INSDC status
public
Submitter Id
E-MTAB-8365:GB1+pJTW100 Rep2
broker name
ArrayExpress
genotype
GB1+pJTW100
growth condition
LB media at 37 degree celsius
organism part
whole organism
sample name
E-MTAB-8365:GB1+pJTW100 Rep2
scientific_name
Escherichia coli str. K-12 substr. MG1655
strain
K-12
substrain
MG1655