Oct 08, 2020

Public workspacefastGRO

DOI
dx.doi.org/10.17504/protocols.io.bbmgik3w
  • 1The Wistar Institute;
  • 2The University of Pennsylvania
Elisa Barbieri
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DOI: dx.doi.org/10.17504/protocols.io.bbmgik3w
Protocol Citation: Elisa Barbieri, Connor Hill, Alessandro Gardini 2020. fastGRO. protocols.io https://dx.doi.org/10.17504/protocols.io.bbmgik3w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2020
Last Modified: October 12, 2020
Protocol Integer ID: 32136
Materials
MATERIALS
Reagent1.5 mL Eppendorf tubes
ReagentChloroform
ReagentIsopropanol
ReagentPBS
ReagentNEBNext Ultra II Directional RNA Library Prep Kit for Illumina - 24 rxnsNew England BiolabsCatalog #E7760S
Reagent0.5M EDTACatalog #AM92606
Reagent2 mL Eppendorf
ReagentGlycerol
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentCorning® 15 ml Centrifuge TubesCorning
ReagentM280 streptavidin beadsInvitrogen - Thermo Fisher
Reagent5M NaClAmbionCatalog #AM9760G
ReagentCapillary electrophoresis instrument (e.g. Agilent Tapestation 4200)
Reagent1M MgCl2 solutionThermo Fisher ScientificCatalog #AM9530G
Reagent50ml Falcon tubesCorningCatalog #352070
ReagentQubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
ReagentTween-20
ReagentEthanol
ReagentKCl 2MCatalog #AM9640G
ReagentSarkosylMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7414
Reagent4-thiouridine (4sU)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T4509
ReagentTRIzol™ LS ReagentThermo FisherCatalog #10296028
ReagentATPThermo FisherCatalog #18330019
ReagentCTPThermo FisherCatalog #18331017
ReagentGTPThermo FisherCatalog #18332015
ReagentEZ-Link™ HPDP-Biotin, No-Weigh™ FormatThermo FisherCatalog #A35390
ReagentSUPERase• In™ RNase Inhibitor (20 U/μL)Thermo FisherCatalog #AM2696
ReagentQubit™ 3 FluorometerThermo FisherCatalog #Q33216
ReagentIGEPAL® CA-630 Merck MilliporeSigma (Sigma-Aldrich)Catalog #I8896
ReagentGlycogenMerck MilliporeSigma (Sigma-Aldrich)Catalog #10901393001
Reagent1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816-10ML
Reagent1M Tris-HCl pH 7.5Thermo Fisher ScientificCatalog #15567027
Reagent1M CaCl2Merck MilliporeSigma (Sigma-Aldrich)Catalog #21115
ReagentNN-DimethylformamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #227056-100ML
Reagent4-Thiouridine-5-Triphosphate (4-thio-UTP)Catalog #N-1025-1
ReagentRNA ScreenTape and ReagentsAgilent Technologies
ReagentBioruptor USD-200Diagenode
Before start
Prepare spike-in RNA or Nuclei
Prepare 50 mM of 4-thiouridine (4sU) in DEPC-treated water. Aliquot and store in the dark at -20 °C.
Incubate drosophila cells for 5 minutes with 50mM of 4sU in their growing medium. Wash cells with 1X PBS, lyse in Trizol reagent. Extract RNA, aliquot, snap-freeze in liquid nitrogen and store at -80 °C.
Can also prepare drosophila nuclei to control for the Nuclear run-on. Can be done using same nuclei extraction protocol (steps 1-9) and drosophila nuclei can be added to your sample at steps 8 or 12 to 5-10% of amount of nuclei from your sample.


Prepare buffers and solutions.

Swelling Buffer (SB) - Add 2 U/ml Superare-In before use.
  • 10 mM Tris-HCL pH 7.5
  • 2 mM MgCl2
  • 3 mM CaCl2
Store at 4 °C.

Swelling Buffer + 10% Glycerol (GSB) - Add 2 U/ml Superare-In before use. Store at 4 °C.

Lysis Buffer (LyB) - Add 2 U/ml Superare-In before use.
  • 10 mM Tris-HCL pH 7.5
  • 2 mM MgCl2
  • 3 mM CaCl2
  • 10% Glycerol
  • 1% Igepal
Store at 4 °C.

Freezing Buffer (FB) - Add 2 U/ml Superare-In before use.
  • 40% glycerol
  • 5 mM MgCl2
  • 0.1 mM EDTA
  • 50 mM Tris-HCL pH8
Store at 4 °C.

1 mg/mL EZ-link HPDP Biotin
Resuspend 1 mg in 1 ml of DMF in polypropylene tubes, vortex and incubate at 36 °C for 30 min.
Store at -20 °C.

10x Biotinylation Buffer
100 mM Tris pH 7.5
10 mM EDTA pH 8.0
Store at 4 °C.
Nuclei isolation
Nuclei isolation
Harvest cells and wash in cold 1X PBS
Resuspend cells in Amount10 mL of ice-cold SB.
Incubate for Duration00:05:00 .
Spin Centrifigation400 x g, 00:10:00 .

Remove supernatant and resuspend in Amount10 mL GSB

Note
Volume of GSB should be at least 5 times the volume of cell pellet

Vortex lightly while adding Amount10 mL of LyB

Incubate on ice for Duration00:05:00
Add Amount25 mL of LyB and centrifuge Centrifigation600 x g, 00:05:00 .

Flick to loosen pellet and resuspend in Amount25 mL of LyB.
Centrifuge Centrifigation600 x g, 00:05:00

Remove supernatant and resuspend in Amount10 mL of FB.
Take Amount10 µL for cell count.

Centrifuge Centrifigation900 x g, 00:06:00 and resuspend using wide-end pipette tips in FB to a concentration of 2x10^7 nuclei per 10 μl of FB.

Nuclei can be stored at Temperature-80 °C for months.

Nuclear Run On
Nuclear Run On

Prepare fresh 2x Nuclear run-on buffer (NRO). (Amount100 µL /sample)
  • 10 mM Tris-HCl ph8
  • 5 mM MgCl2
  • 300 mM KCl
  • 1 mM DTT
  • 500 μM ATP
  • 500 μM GTP
  • 500 μM 4-thio-UTP
  • 2 μM CTP
  • 200 μ/ml Superase-in
  • 1% Sarkosyl (N-Laurylsarcosine sodium salt solution)

Note
Per library, use 1.5-2 x10^7 nuclei


Warm the NRO buffer at Temperature30 °C .



Thaw nuclei TemperatureOn ice .
Note
5-10% drosophila nuclei can be added to your sample as spike-in if not using 4S-U labelled drosophila RNA in step 26.

Mix Amount100 µL of thawed nuclei solution with Amount100 µL of 2xNRO buffer.
Pipette up and down 15 times using end-cut pipette tip.

Incubate Duration00:07:00 at Temperature30 °C .


Add Amount600 µL Trizol LS.
Vortex.
Incubate Duration00:05:00 at TemperatureRoom temperature .

Note

STOP POINT: Freeze with liquid nitrogen, and store at -80 °C

Total RNA precipitation
Total RNA precipitation
Add Amount160 µL of chloroform, shake vigorously by hand for Duration00:00:15

Incubate Duration00:02:00 at TemperatureRoom temperature .

Centrifuge at Centrifigation12000 rpm, 4°C, 00:15:00 .

Transfer upper, aqueous phase into new 1.5 mL centrifuge tube.
Add Amount400 µL of isopropanol to precipitate RNA and incubate at TemperatureRoom temperature for Duration00:10:00 .
Note
Can add 1-2 ul of 2 μg/μL glycogen to allow for visualization of pellet with lower RNA concentrations.

Centrifuge at Centrifigation12000 rpm, 4°C, 00:10:00 .

Wash RNA pellet using Amount1 mL of cold 75 % ethanol
Centrifuge at Centrifigation12000 rpm, 4°C, 00:10:00 .
Completely remove ethanol and air-dry pellet.
Dissolve in Amount100 µL of nuclease-free water.

Determine concentration by Nanodrop or Qubit.
RNA fragmentation
RNA fragmentation
Transfer Amount150 µg of RNA to a 1.5 ml tube and add water up to Amount500 µL .

Note
Save 5 μl of unfragmented RNA to be run on TapeStation as a control for fragmented RNA and to check quality of RNA.


Add 5-10% of labelled spike-in RNA if using instead of drosophila nuclei.
Fragment RNA using Bioruptor with the following settings using: 1 cycle: 30 sec / 30 sec ON / OFF at high settings.

Transfer fragmented RNA to 2 ml tube.
Analyze fragmentation efficiency of fragmented versus unfragmented RNA on Agilent 2200 TapeStation.
Sonicated RNA can be snap-frozen in liquid nitrogen and stored at Temperature-80 °C

EZ-link HPDP-Biotinylation
EZ-link HPDP-Biotinylation

Transfer Amount150 µg of fragmented RNA in one 2 ml Eppendorf tube.
Note
Use only polypropylene tubes during biotinylation.

Incubate RNA at Temperature65 °C for Duration00:10:00 , then TemperatureOn ice for Duration00:05:00 .

Prepare Biotin-RNA mix in 2 ml tube. Follow the order:
  • up to 150 μg of fragmented RNA in Amount500 µL
  • Amount100 µL Biotinylation Buffer
  • Amount200 µL DMF
  • Amount200 µL EZ-link HPDP Biotin



Incubate in the dark at Temperature24 °C and Centrifigation800 rpm for Duration02:00:00 .

Precipitation of biotinylated RNA
Precipitation of biotinylated RNA
Add appr. Amount800 µL of chloroform to the RNA-biotin in the 2 mL phase-lock tube and mix by manually shaking the tube.




Centrifuge at Temperature4 °C and Centrifigation16000 x g Duration00:05:00

Transfer upper phase into new tube (appr 1 mL).

Add 1/10 volume (100 μl) of 5 M NaCl and mix.
Note
If needed at 1-2 μL of 2 μg/μL Glycogen to allow for visualization of pellet with lower RNA concentrations.



Add 1 volume (1 ml) of isopropanol and mix for Duration00:00:15 manually.

Centrifuge Centrifigation16000 x g, 4°C, 00:05:00 .

Remove supernatant.
Wash pellet with Amount1 mL of ice-cold 75% ethanol.
Centrifuge Centrifigation10006 x g, 4°C, 00:30:00 .

Remove supernatant.

Spin quickly at Temperature4 °C and remove remaining supernatant with 200 μl and 10 μl pipettes.
Note
Biotinylated RNA should NOT dry.


Resuspend RNA in Amount100 µL of nuclease-free water.

Note
Biotinylated RNA can be stored at Temperature-80 °C .

Enrichment of biotinylated RNA
Enrichment of biotinylated RNA
Prepare Wash Buffer (WB):
  • 100 mM Tris pH 7.5
  • 10 mM EDTA pH 8.0
  • 1M NaCl
  • 0.1% (vol/vol) Tween-20

Leave half volume of WB atTemperatureRoom temperature and heat the other half at Temperature65 °C .

Prepare the M280 Streptavidin Dynabeads:

Take Amount100 µL of beads per sample.


Wash the beads twice with 2 volumes (Amount200 µL per sample) of wash buffer.


Resuspend in 1 Volume (Amount100 µL per sample) of wash buffer.

Increase the volume of the solution of RNA-biotin to Amount200 µL with nuclease-free water.
Note
Can also scale down to 100 μL if you have less than 150 μg and use 50uL of beads.




Incubate at Temperature65 °C for Duration00:10:00 .
Place on ice for Duration00:05:00 .

Add Amount100 µL of prepared Invitrogen streptavidin beads to Amount200 µL of RNA-biotin.

Incubate atTemperature4 °C for Duration00:15:00 in rotation.

Transfer tubes to a magnetic rack.
Remove supernatant.
Do not disturb beads.
Wash at least 3 times with Amount900 µL of warm (Temperature65 °C ) WB.

Wash at least 3 times with Amount900 µL of room temperature WB.

Resuspend beads in in Amount100 µL of 100 mM DTT and incubate Duration00:05:00 .

Transfer tubes to the magnetic rack.
Collect the 4-thio-labeled RNA in a new tube.
Repeat steps 57-58 and collect the eluted RNA in the same tube (Amount200 µL total volume).

Purification of labelled RNA with RNA Clean and
Purification of labelled RNA with RNA Clean and
Use buffers provided with the RNA Clean and Purification kit-5 (Zymo Research). Add ethanol to wash and pre-wash buffers and resuspend DNAse in water.



Add Amount400 µL of RNA Binding Buffer to each sample and mix.

Add Amount600 µL of ethanol (95-100%) and mix.


Transfer the sample to the Zymo-Spin IC Column in a Collection Tube and centrifuge for Duration00:00:30 . Discard the flow-through.


Add Amount400 µL of RNA Wash Buffer to the column and centrifuge for Centrifigation16000 x g, 00:00:30 . Discard the flow-through.

For each sample to be treated, prepare DNase I reaction mix in an RNase-free tube. Mix well by gentle inversion:
  • Amount5 µL DNase I
  • Amount35 µL DNA Digestion Buffer

Add Amount40 µL reaction mix directly to the column matrix. Incubate at TemperatureRoom temperature forDuration00:15:00 .

Add Amount400 µL RNA Prep Buffer to the column and centrifuge for Duration00:00:30 . Discard the flow-through.

Add Amount700 µL RNA Wash Buffer to the column and centrifuge for Duration00:00:30 . Discard the flow-through.

Add Amount400 µL RNA Wash Buffer to the column and centrifuge for Duration00:02:00 . Discard the flow-through.

Centrifuge for Duration00:01:00 at full speed to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.

Add Amount6 µL DNase/RNase-Free Water directly to the column matrix, incubate for Duration00:01:00 and centrifuge for Duration00:00:30

Measure concentration of labelled RNA by Qubit fluorometer.
Libraries can be prepared with NEBNext Ultra II Directional RNA Library Prep or other library prep kits.