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Kane 10X prion handling experiment

Kane 10X prion handling experiment

Overview

1 uninfected pool of splenocytes provided by Sarah Kane will be single cell sorted and have barcoded cDNA synthesized. 

This will be split into three (perhaps with portion one getting 3/5ths).  The second portion will be exposed to NaOH for an hour.  The third portion will go through ssDNA cleanup.  All portions will be VDJ amplified.  The untreated perhaps larger portion would then be split into thirds with one getting no treatment/cleanup, one  treated with NaOH for an hour, and the third being treated with an Amplicon cleanup kit.  All 5 groups would then proceed normally through the 10X procedures and be checked on the tapestation and via qPCR.

Kane Steps

Fix and quench cells

Probe Hybridization

Thaw Hyb Buffer B @42C keep warm vortex and quick spin
Thaw Enhancer @65C keep warm vortex and quick spin
Thaw Appropriate Probes on ice
Incubate Hybridization Mix at 42C for 5 minutes:

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

Hyb B

70

 

 

Enhancer

10

 

 

Centrifuge 50,000 - 2,000,000 cells per reaction in Quenching Buffer at 850 rcf for 5 minutes
Remove Supernatant
Resuspend with 80 ul Hybridization Mix
Add 20 ul Appropriate Probe
Incubate in prewarmed thermocycler or incubator at 42 C with heated lid for 16-24 hours

Gem Generation and barcoding

Prepare Reagents:
Thaw at RT Single Cell TL Gel Beads and Reducing Agent B
Thaw at 65C for >10m Enhancer
Thaw on Ice Conc Post-Hyb Buffer, GEM Enzyme Buffer, and GEM Reagent Mix
Set out at RT Partitioning Oil, Next GEM Chip, Chip holder, gasket, Sample filters, 10X vortex adapter, 50% glycerol, Glycerol
Prepare Post-Hyb Wash Buffer and keep at RT

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

H2O

1800

 

 

Conc Post Hyb

100

 

 

Enhancer

100

 

 

Total

2000

 

 

Add 900 ul Post-Hyb Wash Buffer to each tube and Pipette Mix 5X
Centrifuge 850 rcf for 5m and remove supernatant
Resuspend pellets in 500 ul Post-Hyb Wash Buffer
Incubate at 42 C for 10m
Centrifuge 850 rcf for 5m and remove supernatant
Resuspend pellets in 500 ul Post-Hyb Wash Buffer
Incubate at 42 C for 10m
Prepare PostHyb Resuspension Buffer and maintain at 4C:

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

H20

475

 

 

Conc Post Hyb

25

 

 

Centrifuge sample at 850 rcf for 5m and remove supernatant
Resuspend in appropriate amount of PostHyb Resuspension Buffer
Pipette Mix 20x
Pass the sample through a 30 micron filter
Measure cell concentration
Store sample at -80 for up to 6 months by
Add 0.1 V Enhancer
Add 50% glycerol to a final conc of 10%
Obtain Chromium Chip
Dilute to appropriate concentration
Add 35 ul GEM MM and immediately proceed to next step

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

GEM reagent Mix

20.9

 

 

Reducing Agent B

1.7

 

 

GEM Enzyme Mix

12.4

 

 

Add 50% glycerol to all unused wells
b.Prepare Gel Beads
lSnap the tube strip holder with the Gel Bead strip into a
10x Vortex Adapter. Vortex 30 sec.
Centrifuge the Gel Bead strip for ~5 sec.
Confirm there are no bubbles at the bottom of the tubes & theliquid levels are even.
Place the Gel Bead strip back in the holder. Secure the holder lid.
Load Row 1
With pipette set to 70 μl, gently pipette mix the GEM Master Mix 15x.
Using the same pipette tips, dispense 70 μl GEM Master Mix +Sample into the bottom center of wells in row labeled 1 without introducing bubbles.
Load Row 2
Add 50 ul Gel Beads slowly
Load Row 3
Add 45 ul Partitioning Oil
Close the Chip lid
Run the chip on the Chromium X
Slowly Transfer 100 ul from Recovery wells to tube strip on ice

Post GEM Recovery

Add 125 ul Recovery Agent at RT
Mix by inversion 5x
Wait 2 minutes
Centrifuge briefly
Slowly Remove 125 ul Recovery Agent/Partitioning Agent (pink) from bottom of tube
Proceed to Pre-Amplification PCR

Pre-Amp PCR

Prepare Pre-Amp Mix on ice, vortex and briefly centrifuge

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

Amp Mix

25

 

 

Pre-Amp Primers B

10

 

 

Add 35 ul to aqueous sample
Invert 8x to mix, centrifuge briefly
Add to Thermocycler

Temp

Time

Cycles

Temp

Time

Cycles

98

3:00

1x

98

0:15

8x

63

0:20

72

1:00

72

1:00

1x

8

hold

1x

Bead Cleanup

Prepare Elution solution

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

Buffer EB

980

 

 

10% Tween20

10

 

 

Reducing Agent B

10

 

 

Centrifuge PCR product 30 s and transfer 70 ul upper layer to new tube
Add 126 ul Mag Beads to each sample and pipette mix 15x
Incubate at RT 5 min
Incubate on magnet 5 min
Remove supernatant
Add 200 ul 80% EtOH
Incubate 30s, then remove EtOH
Add 200 ul 80% EtOH
Incubate 30s, then remove EtOH x2
Dry 2 minutes
Remove from magnet and add 101 ul Elution solution
Incubate 1 minute, then resuspend via pipette mix 15x
Incubate 2 minutes
Incubate 2 minutes on magnet
Transfer 100 ul to new tubes

Sample Index PCR

Add 60 ul MM to each

Reagent

ul for 1

Rxns (x1.1)

ul needed

Reagent

ul for 1

Rxns (x1.1)

ul needed

Amp Mix

50

 

 

H2O

10

 

 

Run on Thermocycler