Notebook

Buffers To Make¶

We will use the Same Recipe as Zilionis et. al (2016) Filter all These Buffers Before use.

TE-SDS:
- 10 mM Tris HCl pH 8.0 + 1 mM EDTA
- 0.5% SDS
TE-TW:
- 10 mM Tris HCl pH 8.0 + 1 mM EDTA
- 0.01% Tween-20
Hybridization Buffer:
- 10mM Tris-HCl pH 8.0
- 0.1mM EDTA pH 8.0
- 0.1% Tween-20
- 330 mM KCl
IDTE Buffer(Buffer for Disolving Oligos Ordered from IDT):
- 10 mM Tris HCl pH 8.0 + 0.1mM EDTA

Protocol for Exo1 Digestion and Removal of Primer from Beads for ChronoSeqV2 Beads¶

Get the Exonuclease 1 Buffer -20°C and leave on your bench to melt. Put on ice once melted.
Get the Exonuclease 1 from -20°C and put it on ice.
Make 1mM stocks for the Oligos orders from IDT using the IDTE buffer.
Set the Fisher Brand Heater Cooler to 80°C and 1000RPM. Start the program to preheat to 80°C
Set an incubator to 37°C
Turn on the UV and airflow for the Cell Culture Hood.
Spin Down S9,S10,S11 and S12 Tubes with 450beads/μl suspended in 1X Lysis buffer.
Shake Each Tube to make sure the beads are evenly suspended . For each Tube using a Sterile/Particle Free Rainin P1000 pipette tip transfer 100μl of beads to a Sterile 1.5ml Eppendorf Tube. After you have transfered 100μl from each of the 4 tubes you will have 400μl of beads @ 450beads/μl in the 1.5ml Eppie.
Spin the Beads down and remove 300μl of Lysis buffer. Without disturbing the beads.
Now wash the beads 3 Times with 1ml Hybridization Buffer. Spin down each time.
Remove 500μl of Buffer. Then Estimate the Volume and make the Final Volume of the Beads 350μl.
Add 20μl of 1mM A30 or BA19 Oligo.
Heat this mixture upto 80°C Using the Fisher Brand Heater Cooler or Water Bath at 1000RPM.
Heat for 5 minutes once the Heater/Cooler has reached the Required Temperature.
Remove from the Heater Cooler and then leave at Room Temperature for 5 minutes.
Spin down the beads and add:
1. 100μl of Exonuclease 1 buffer (10X).
2. Now add 480μl of Distilled Water.
3. 50μl of Exonuclease 1.
Vortex the Beads to Mix everything Well.
Now Incubate at 37°C with Rotation for 2.5 hours.
Set the incubator back to 42°C after you are done using it.
Spin Down the Beads.
Remove 900μl of Buffer.
Add 1000μl of TE-SDS.
Vortex the beads and shake them by hand as well.
Spin down the Beads and wash twice with 1ml of TE-TW.

THIS IS A STOPPING STEP. You can store the Beads in TE-TW at 4°C and continue later.

Wash again Twice with 2ml of Distilled Water.
Remove 1ml of Distilled Water from the Beads after Spining Down. There should be about 100μl liquid left inside the Eppie.

Protocol for Removal of Primer using Sodium Hydroxide Alkaline Wash.¶

Sodium Hydroxide is a very dangerous Chemical it can melt your Skin and turn your body into Soap. You need to be extra careful.
Wear Lab Coat, Safety Glasses, Apron, Face-Sheild, Double Nitrile Gloves and work in the Fume Hood.
Make sure you don't have exposed skin by covering your gloves with the apron.
Replace the work area mats with Fresh new ones.
Make sure there is enough space to work inside the Fume hood.
Bring the 10M NaOH Bottle stored under your bench in a secondary container to the Fume Hood. You should always store the bottle inside a secondary container inside another larger container.
You will need one Hazardous Waste Container for the Tips. Bring The Black Container into The Fume Hood. Check the Waste Tag to make sure this is the correct container.
You will also need a Buffer to Dispose off the 1M NaOH washed through.
For this purpose I recommend having 40ml of 1M Tris-HCl pH 7.5 in a 50ml Tube Ready.
Set aside another 5ml of 1M Tris-HCl pH 7.5 in a different tube for washing the Beads.
You will also need to prepare a Spray Bottle full of 1M Tris-HCl pH 7.5 Buffer. This will act as our emergency spray and cleaning things after we are done. Label This bottle correctly and Keep it at your bench or inside the Fume Hood. Very important to have this with you.
We Will now make Fresh Denaturation Solution. To Make 2ml of Denaturation Solution we will need the Following:
1. 1800μl of Distilled Water (Add 1000μl then 800μl using P1000)
2. 200μl of 10M NaOH.
Wash the P1000 Tips into 1M Tris-HCl Buffer and deposit the waste Denaturation Solution into the Buffer.
Now Safetly Discard these Tips into the Black Hazardous Waste Bin.
Put the 10M NaOH back in its storage location inside its secondary container.
Change your gloves.
Now Carefully Add 1ml of Denaturation Solution. and Vortex the Beads to Mix Well.
Discard the Tip into the Black Bin.
Close the Eppie.

OPTIONAL in case of a Spill: Spray the Exterior of the Eppie with 1M Tris-HCl and wipe the Eppie dry with Paper wipes. Spray the Gloves as well and then Dry them using Paper Wipes.

Vortex the beads. Then, leave the Beads for 1 minute.
Now Spin down the Beads.
Remove 1ml of the Denaturation Solution and Deposit it into the 40ml Tris-HCl you set aside earlier.
Now Discard the Tip into the Black Bin.
Wash one more time with 1ml Denaturation Solution using the same steps as above.
Now wash Twice with 1ml 1M Tris-HCl. Discard the buffer into the 40ml Tris-HCl tube. Discard the Tip into the Black Bin each time.

OPTIONAL in case of a Spill: Spray the bottles surfaces, eppies, gloves, facesheild and/or apron with Tris-HCl and dry with paper towels.

Make sure the 10M NaOH bottle is closed tightly. Store it in its correct location. Also store the black bin.
Remove the apron and store for later use.
You can now move back to your bench from the fume hood.
Wash the beads thrice with 1ml 1X Lysis Buffer.
Remove 300μl of Lysis buffer. Now Count the Beads to get the Bead Concentration.
Make Final Concentration of 450beads/μl.
Follow the Bulk RNA Seq protocol to verify your beads are working or not.