TITLE: fastGRO
AUTHORS: Elisa Barbieri, Connor Hill, Alessandro Gardini

[STEPS]
?. [Nuclei isolation]
Harvest cells and wash in cold 1X PBS
?. [Nuclear Run On]
Prepare fresh 2x Nuclear run-on buffer (NRO). (/sample)10 mM Tris-HCl ph8 5 mM MgCl2300 mM KCl1 mM DTT 500 μM ATP500 μM GTP500 μM 4-thio-UTP2 μM CTP200 μ/ml Superase-in1% Sarkosyl (N-Laurylsarcosine sodium salt solution)
100 µl
Per library, use 1.5-2 x10^7 nuclei
?. [Nuclei isolation]
Centrifuge   and resuspend using wide-end pipette tips in FB to a concentration of 2x10^7 nuclei per 10 μl of FB.
Centrifuge: 900 34, 6 min
?. [Nuclei isolation]
Nuclei can be stored at  for months.
-80 °C
?. [Nuclei isolation]
Remove supernatant and resuspend in  of FB. Take  for cell count.
10 mL
10 µl
?. [Nuclei isolation]
Flick to loosen pellet and resuspend in of LyB. Centrifuge
25 mL
Centrifuge: 600 34, 5 min
?. [Nuclei isolation]
Incubate on ice for Add  of LyB and centrifuge  .
25 mL
Centrifuge: 600 34, 5 min
?. [Nuclei isolation]
Vortex lightly while adding  of LyB
10 mL
?. [Nuclei isolation]
Remove supernatant and resuspend in  GSB
10 mL
Volume of GSB should be at least 5 times the volume of cell pellet
?. [Nuclear Run On]
Warm the NRO buffer at .
30 °C
?. [Total RNA precipitation]
Add  of chloroform, shake vigorously by hand for
160 µl
?. [Total RNA precipitation]
Incubate  at  .
0 Room temperature
?. [Total RNA precipitation]
Centrifuge at  .
Centrifuge: 12000 33, 15 min, 4 10
?. [Total RNA precipitation]
Transfer upper, aqueous phase into new 1.5 mL centrifuge tube.
?. [Total RNA precipitation]
Add  of isopropanol to precipitate RNA and incubate at   for  .
400 µl
0 Room temperature
Can add  1-2 ul of 2 μg/μL glycogen to allow for visualization of pellet with lower RNA concentrations.
?. [Total RNA precipitation]
Centrifuge at   .
Centrifuge: 12000 33, 10 min, 4 10
?. [Total RNA precipitation]
Wash RNA pellet using  of cold 75 % ethanolCentrifuge at   .
1 mL
Centrifuge: 12000 33, 10 min, 4 10
?. [Total RNA precipitation]
Completely remove ethanol and air-dry pellet. Dissolve in  of nuclease-free water.
100 µl
?. [Total RNA precipitation]
Determine concentration by Nanodrop or Qubit.
?. [Nuclei isolation]
Resuspend cells in  of ice-cold SB. Incubate for . Spin .
10 mL
Centrifuge: 400 34, 10 min
?. [Nuclear Run On]
Mix  of thawed nuclei solution with  of 2xNRO buffer. Pipette up and down 15 times using end-cut pipette tip.
100 µl
100 µl
?. [Nuclear Run On]
Incubate   at   .
30 °C
?. [Nuclear Run On]
Add  Trizol LS. Vortex. Incubate   at .
600 µl
0 Room temperature
STOP POINT: Freeze with liquid nitrogen, and store at -80 °C
?. [Nuclear Run On]
Thaw nuclei  .
on ice
5-10% drosophila nuclei can be added to your sample as spike-in if not using 4S-U labelled drosophila RNA in step 26.
?. [RNA fragmentation]
Transfer  of RNA to a 1.5 ml tube and add water up to .
150 µg
500 µl
Save 5 μl of unfragmented RNA to be run on TapeStation as a control for fragmented RNA and to check quality of RNA.
?. [EZ-link HPDP-Biotinylation]
Transfer  of fragmented RNA in one 2 ml Eppendorf tube.
150 µg
Use only polypropylene tubes during biotinylation.
?. [Precipitation of biotinylated RNA]
Add appr. of chloroform to the RNA-biotin in the 2 mL phase-lock tube and mix by manually shaking the tube.
800 µl
?. [Enrichment of biotinylated RNA]
Prepare Wash Buffer (WB):100 mM Tris pH 7.5 10 mM EDTA pH 8.01M NaCl0.1% (vol/vol) Tween-20
?. [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.
?. [RNA fragmentation]
Add 5-10% of labelled spike-in RNA if using instead of drosophila nuclei.
?. [RNA fragmentation]
Fragment RNA using Bioruptor with the following settings using: 1 cycle: 30 sec / 30 sec ON / OFF at high settings.
?. [RNA fragmentation]
Transfer fragmented RNA to 2 ml tube.
?. [RNA fragmentation]
Analyze fragmentation efficiency of fragmented versus unfragmented RNA on Agilent 2200 TapeStation.
?. [RNA fragmentation]
Sonicated RNA can be snap-frozen in liquid nitrogen and stored at
-80 °C
?. [EZ-link HPDP-Biotinylation]
Incubate in the dark at   and   for  .
24 °C
Centrifuge: 800 33
?. [Purification of labelled RNA with RNA Clean and ]
Libraries can be prepared with NEBNext Ultra II Directional RNA Library Prep or other library prep kits.
?. [Purification of labelled RNA with RNA Clean and ]
Measure concentration of labelled RNA by Qubit fluorometer.
?. [Enrichment of biotinylated RNA]
Repeat steps 57-58 and collect the eluted RNA in the same tube ( total volume).
200 µl
?. [EZ-link HPDP-Biotinylation]
Prepare Biotin-RNA mix in 2 ml tube. Follow the order:up to 150 μg of fragmented RNA in  Biotinylation Buffer DMF EZ-link HPDP Biotin
500 µl
100 µl
200 µl
200 µl
?. [EZ-link HPDP-Biotinylation]
Incubate RNA at   for  , then   for  .
65 °C
on ice
?. [Enrichment of biotinylated RNA]
Transfer tubes to the magnetic rack.Collect the 4-thio-labeled RNA in a new tube.
?. [Enrichment of biotinylated RNA]
Resuspend beads in in of 100 mM DTT and incubate   .
100 µl
?. [Enrichment of biotinylated RNA]
Wash at least 3 times with of room temperature WB.
900 µl
?. [Enrichment of biotinylated RNA]
Wash at least 3 times with  of warm () WB.
900 µl
65 °C
?. [Enrichment of biotinylated RNA]
Transfer tubes to a magnetic rack.Remove supernatant.Do not disturb beads.
?. [Enrichment of biotinylated RNA]
Incubate at for  in rotation.
4 °C
?. [Enrichment of biotinylated RNA]
Add of prepared Invitrogen streptavidin beads to  of RNA-biotin.
100 µl
200 µl
?. [Enrichment of biotinylated RNA]
Increase the volume of the solution of RNA-biotin to  with nuclease-free water.
200 µl
Can also scale down to 100 μL if you have less than 150 μg and use 50uL of beads.
?. [Enrichment of biotinylated RNA]
Incubate at  for . Place on ice for .
65 °C
?. [Enrichment of biotinylated RNA]
Prepare the M280 Streptavidin Dynabeads:
?. [Enrichment of biotinylated RNA]
Take  of beads per sample.
100 µl
?. [Enrichment of biotinylated RNA]
Wash the beads twice with 2 volumes (per sample) of wash buffer.
200 µl
?. [Enrichment of biotinylated RNA]
Resuspend in 1 Volume ( per sample) of wash buffer.
100 µl
?. [Enrichment of biotinylated RNA]
Leave half volume of WB at and heat the other half at .
0 Room temperature
65 °C
?. [Precipitation of biotinylated RNA]
Resuspend RNA in of nuclease-free water.
100 µl
Biotinylated RNA can be stored at  .
?. [Precipitation of biotinylated RNA]
Spin quickly at  and remove remaining supernatant with 200 μl and 10 μl pipettes.
4 °C
Biotinylated RNA should NOT dry.
?. [Precipitation of biotinylated RNA]
Remove supernatant.
?. [Precipitation of biotinylated RNA]
Wash pellet with  of ice-cold 75% ethanol.Centrifuge  .
1 mL
Centrifuge: 10006 34, 30 min, 4 10
?. [Precipitation of biotinylated RNA]
Remove supernatant.
?. [Precipitation of biotinylated RNA]
Centrifuge  .
Centrifuge: 16000 34, 5 min, 4 10
?. [Purification of labelled RNA with RNA Clean and ]
Add  DNase/RNase-Free Water directly to the column matrix, incubate for  and centrifuge for
6 µl
?. [Purification of labelled RNA with RNA Clean and ]
Centrifuge for  at full speed to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
?. [Precipitation of biotinylated RNA]
Add 1 volume (1 ml) of isopropanol and mix for  manually.
?. [Precipitation of biotinylated RNA]
Add 1/10 volume (100 μl) of 5 M NaCl and mix.
If needed at 1-2 μL of 2 μg/μL Glycogen to allow for visualization of pellet with lower RNA concentrations.
?. [Precipitation of biotinylated RNA]
Transfer upper phase into new tube (appr 1 mL).
?. [Precipitation of biotinylated RNA]
Centrifuge at   and
4 °C
Centrifuge: 16000 34
?. [Purification of labelled RNA with RNA Clean and ]
Add  RNA Wash Buffer to the column and centrifuge for . Discard the flow-through.
400 µl
?. [Purification of labelled RNA with RNA Clean and ]
Add  RNA Wash Buffer to the column and centrifuge for . Discard the flow-through.
700 µl
?. [Purification of labelled RNA with RNA Clean and ]
Add  RNA Prep Buffer to the column and centrifuge for . Discard the flow-through.
400 µl
?. [Purification of labelled RNA with RNA Clean and ]
Add reaction mix directly to the column matrix. Incubate at  for.
40 µl
0 Room temperature
?. [Purification of labelled RNA with RNA Clean and ]
Add  of  RNA Binding Buffer to each sample and mix.
400 µl
?. [Purification of labelled RNA with RNA Clean and ]
Add  of ethanol (95-100%) and mix.
600 µl
?. [Purification of labelled RNA with RNA Clean and ]
Transfer the sample to the Zymo-Spin IC Column in a Collection Tube and centrifuge for . Discard the flow-through.
?. [Purification of labelled RNA with RNA Clean and ]
Add  of RNA Wash Buffer to the column and centrifuge for . Discard the flow-through.
400 µl
Centrifuge: 16000 34, 30 s
?. [Purification of labelled RNA with RNA Clean and ]
For each sample to be treated, prepare DNase I reaction mix in an RNase-free tube. Mix well by gentle inversion:  DNase I  DNA Digestion Buffer
5 µl
35 µl