title: CFZ 2
organism: Mycobacterium tuberculosis
description: Protocols: 1. Mtb cell pellet collection • Pour culture into 2x volume GTC+βME and incubate for 5 minutes in the dark at room temperature - example: 1 ml culture + 2 ml GTC+ βME - if larger than 15 ml total culture, will need multiple tubes for spinning down • Centrifuge for 5 minutes at 4000 rpm and dump supernatant (GTC waste must be kept separate from general culture waste and taken out of the BSL3 without autoclaving) • Can flash freeze on dry ice and store at -80°C; resuspend in 1 ml Trizol and store at -80°C; or proceed directly to RNA isolation 2. RNA Isolation - Bead-Beating and Trizol Extraction • Add 1 ml Trizol to the pellet (1 ml per 50 ml mid-log culture) while on ice and resuspend pellet using pipette • Transfer Trizol suspension to a 2.0 ml screw cap tube containing 0.4 ml of 0.1 mm silica beads • Disrupt Mtb cells for 40 sec at speed 48 (BeadBug: 40 sec, 4000 rpm), 3x. Cool on ice between cycles (cool for 3-5 minutes minimum between cycles) • Continue to periodically invert tubes for 5 minutes on ice • Centrifuge for 1 min at max speed (15000 rpm) • Transfer supernatant to a 2.0 ml screw cap tube containing heavy phase lock gel (if doing DNA back-extraction do not use phase lock gel). Add 300 μl chloroform (200 ul if not using phase lock). • Invert tube vigorously for 15 sec. Continue to invert tube for 2 min. • Centrifuge for 10 min at max speed. • Transfer aqueous phase (~540 μl per 1 ml Trizol) to a 2.0 ml elution tube containing 270 ul isopropanol, mix well by inversion • Add 265 ul high salt solution (0.8 M sodium citrate, 1.2 M NaCl), mix well by inversion. • Spray off tubes and remove from BSL3 • Incubate at 4°C overnight • Next day, centrifuge at 4°C for 10 minutes, max speed • Aspirate isopropanol. Wash twice in 0.5 ml 70% ethanol, spinning for 5 min after each wash (can be at RT) 3. DNase treatment and column purification • Remove trace ethanol by speedvac (3 min spin), reconstitute by adding 90 μl RNase-free water (if pellet will not go into solution, can heat at 37°C) - if combining multiple tubes of same sample, do it here Make sure that all liquid is gone, pellet should be dry (can also air-dry, making sure pellet is actually dry before reconstitution) • Once pellet is reconstituted, add 10 μl Roche DNase buffer and 1 μl DNase. Mix by gently flicking side of tube. • Incubate in 37°C water bath for 30 min • Add 350 μl fresh RLT buffer + 10μl/ml βME to sample and vortex thoroughly to mix. Add 250 μl 100% EtOH and invert to mix. • Add sample (total volume should be ~ 700 μl) to new RNeasy column and centrifuge at max speed for 30 sec • Wash column by adding 700 μl of buffer RW1 to RNeasy column and centrifuge for 30 sec. Discard eluate. Repeat wash with 700 μl of buffer RW1 and centrifuge a second time for 30 sec • Transfer RNeasy column to a new collection tube. Wash by adding 500 μl buffer RPE (ensure that EtOH has been added to this buffer) to RNeasy column and centrifuge for 30 sec. Discard eluate. Repeat wash with 500 μl of RPE and centrifuge a second time for 2 min. Discard eluate. • Spin column in empty collection tube for 1 min to dry column membrane • Transfer column to an RNase-free 1.5 ml Eppendorf tube (from kit), open cap of column and allow membrane to air dry for 2 min. • Elute sample by adding 50 μl of RNase-free water directly to the center of the membrane (do not want any water on the sides of the tube). Do not close cap, let membrane soak for 2 min. Close cap and centrifuge for 2 min. Perform second elution in the same collection tube with 44μl of RNase-free water. Total sample volume should be 90 μl (eluate is RNA). • Add 10 μl Roche DNase buffer and 1 μl DNase to eluted RNA. Mix by gently flicking side of tube. • Incubate in 37°C water bath for 30 min • Add 350 μl fresh RLT buffer + 10μl/ml βME to sample and vortex thoroughly to mix. Add 250 μl 100% EtOH and invert to mix. • Add sample (total volume should be ~ 700 μl) to new RNeasy column and centrifuge at max speed for 30 sec • Wash column by adding 500 μl buffer RPE (ensure that EtOH has been added to this buffer) to RNeasy column and centrifuge for 30 sec. Discard eluate. Repeat wash with 500 μl of RPE and centrifuge a second time for 2 min. Discard eluate. • Spin column in empty collection tube for 1 min to dry column membrane • Transfer column to an RNase-free 1.5 ml Eppendorf tube (from kit), open cap of column and allow membrane to air dry for 2 min. • Elute sample by adding 40 μl of RNase-free water directly to the center of the membrane (do not want any water on the sides of the tube). Do not close cap, let membrane soak for 2 min. Close cap and centrifuge for 2 min. Perform second elution in the same collection tube with the already eluted 40 μl RNA (take liquid from the collection tube and run it through the column again). Centrifuge for 2 minutes. Total sample volume should be 40 μl. • Quantify RNA concentration using Nanodrop. • Store RNA samples at -80°C 100ng of Total RNA using Nugen Universal mRNA Library Construction Kit Cat # 0520.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-07-22T04:06:33Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-07-15T11:20:18Z
attributes.tag: External Id
attributes.value: SAMEA7060545
attributes.tag: INSDC center name
attributes.value: University of Colorado Anschutz School of Medicine, Department of Immunology and Microbiology
attributes.tag: INSDC first public
attributes.value: 2020-07-22T04:06:33Z
attributes.tag: INSDC last update
attributes.value: 2020-07-15T11:20:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9350:CFZ 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: sample name
attributes.value: E-MTAB-9350:CFZ 2
attributes.tag: scientific_name
attributes.value: Mycobacterium tuberculosis
attributes.tag: strain
attributes.value: Erdman TMCC 107