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Murine B-cells PIP-ReAmp 1

Murine B-cells PIP-ReAmp 1

  • ReAmplification from saved PIPs of B1

Obtain frozen PIPs
Combine 300 ul UPW with 300 ul Washing Buffer per sample to make 0.5X Wash
Briefly spin down PIPs
Add 120 ul 0.5 X Wash
Centrifuge 5s add to guide rack
Tap 3x and let settle 1 minute
Aspirate and discard 150 ul (don’t disturb pellet)
Add 150 ul 0.5X Wash
Centrifuge 5s add to guide rack
Tap 3x and let settle 1 minute
Aspirate and discard 150 ul (don’t disturb pellet)
Add 150 ul 0.5X Wash
Centrifuge 5s add to guide rack
Tap 3x and let settle 1 minute
Aspirate and discard 150 ul (don’t disturb pellet)Add 150 ul 0.5X Wash
Centrifuge 5s add to guide rack
Tap 3x and let settle 1 minute
Aspirate and discard 150 ul (don’t disturb pellet)
Reduce V to guide wire

cDNA reAmplification

Pipette mix WTA Primer and centrifuge briefly
Make 1X WTA Primer (3 ul WTA and 27 ul H2O)
Make PCR MM using pipette mixing for reagents

Reagent

V per run

1.1 x

2.2x

8.8x

17.6

Reagent

V per run

1.1 x

2.2x

8.8x

17.6

4X PCR MM

15

16.5

33

132

264

1X WTA Primer

6

6.6

13.2

52.8

105.6

Total

21

23.1

46.2

184.8

369.6

Add 21 ul MM into each PCR tube
Pulse vortex in black stand and quick spin down
Run on Thermocycler with 105 C lid:

Temp

Time

Cycles (60 ul)

Temp

Time

Cycles (60 ul)

95

3:00

1

98

0:15

4

69

10:00

4

72

5:00

1

4

0:00

1

HOLD POINT 4 C in Thermocycler overnight

Isolate cDNA from PIPs in Post PCR area

B1
B2
B3
B4
Stage QC reagents on ice
4X PCR MM
WTA Primer (do not dilute)
Washing Buffer
Stage Reagents at RT
CE Buffer, low EDTA TE, ultra pure water
Illumina Purification Beads (limit light exposure) >20 minutes
0.2 ml tubes or strip tubes and lids
Magnet rack
Add 40 ul CE Buffer to each WTA rxn and seal with new lids
Pulse vortex and spin down
Place in guide rack, tap on bench, and wait > 1 minute for PIPs to settle below guide
Transfer 60 ul supernatant into a NEW labeled 0.2 ml tube
Add another 60 ul CE Buffer to the original tubes
Pulse vortex, spin down 5s, load back into guide rack
Tap on bench and let settle >1 minute
Transfer 60 ul supernatant into tubes containing supernatant
Briefly centrifuge supernatant tubes.
If PIPs are present, transfer supernatant to new tubes
Save remaining PIP pellet at -80C as backup

MagBead Purification

Prepare at least 400 ul of fresh 85% Ethanol per rxn (340 ul EtOH and 60 ul ultra pure water)
Resuspend Illumina Purification beads via vortex <30s
Check Volumes of samples
Add 0.8x beads to each sample (96 ul beads for 120 ul sample or sampleX0.8 of beads)
Vortex until thoroughly mixed then pulse centrifuge to bring liquid to bottom
Incubate 5 min at RT
Place tubes in magnetic stand for 5 minutes or until liquid is clear
Remove supernatant <216 ul
Add 200 ul 85% EtOH, incubate 30 s, and discard
Add 200 ul 85% EtOH, incubate 30 s, and discard
Pipette again to remove all EtOH
Air Dry for 2-5 minutes
Remove from magnet and Add 42 ul low EDTA TE to each sample
Pipette mix 10x or until beads are resuspended
Incubate 5 minutes at RT
Return tubes to magnet for 2 minutes
Remove and save 40 ul of supernatant in new tubes and place on ice

cDNA QC of remaining 2 ul

Add 9 ul ultra pure water to remaining 2 ul and mix without disturbing beads
Incubate 1 minute
Transfer 10 ul to new PCR tubes and place on ice
Prepare QC MM:

Reagent

V per rxn

1.1X

4.4X

8.8X

17.6

Reagent

V per rxn

1.1X

4.4X

8.8X

17.6

4X PCR MM

6.25

6.9

27.6

55

110

WTA Primer

1

1.1

4.4

8.8

17.6

0.5X Washing Buffer

7.75

8.5

34

68.2

136.4

Total

15

16.5

66

132

264

Add 15 ul of QC MM to each 10 ul sample
Run on Thermocycler:

Temp

Time

Cycles (25 ul)

Temp

Time

Cycles (25 ul)

95

3:00

1

98

0:15

13

69

4:00

13

72

5:00

1

4

0:00

1

HOLD POINT 4 C in Thermocycler overnight

QC bead cleanup

B1
B2
B3
B4
Stage Reagents at RT
CE Buffer
Illumina Purification Beads >20 minutes
ultra pure water
low EDTA TE
tapestation supplies
Prepare at least 400 ul of fresh 85% Ethanol per rxn (340 ul EtOH and 60 ul ultra pure water)
Add 15 ul ultra pure water to each sample
Add 32 ul Illumina Purification Beads, Mix by pipette 15 times with 67 ul
Incubate 5 minutes RT
Place on Magnet, Incubate 5 minutes
Discard ~72 ul supernatant
Add 200 ul 85% EtOH, incubate 30 s, and discard
Add 200 ul 85% EtOH, incubate 30 s, and discard
Pipette again to remove all EtOH
Air Dry for 2-5 minutes
Remove from magnet, Add 11 ul low EDTA TE, Mix by pipette >10X
Incubate 5 minutes RT
Return tubes to magnet for 2 minutes
Remove and save 10 ul supernatant for QC
Run samples on HS D5000 ScreenTape

Library Prep

Stage Reagents at RT
Illumina Purification Beads >20 minutes
ultra pure water
low EDTA TE
Thaw Reagents on ice
Library Prep Buffer
Library Prep Enzymes
Library Prep Mix A
Library Adapter Mix
4 X PCR MM
UDI Library Index Mix Strip
Obtain the 40 ul of cDNA and place on ice
Pre-Chill Thermocycler
Add 10 ul MM to each tube:

Reagent

V per sample

2.2

8.8

17.6

Reagent

V per sample

2.2

8.8

17.6

Library Prep Buffer

4

8.8

35.2

70.4

Library Prep Enzymes

6

13.2

52.8

105.6

Vortex to mix 5-10s
Thermocycle with lid at 105C:

Temp

Time. (50ul)

Temp

Time. (50ul)

4

0:00

30

6:00

65

30:00

4

0:00

Prepare Library Adapter Mix

Reagent

V per rxn

2.2

8.8

17.6

Reagent

V per rxn

2.2

8.8

17.6

Library Adapter Mix

0.75

1.7

6.6

13.2

Ultra Pure Water

4.25

9.3

37.4

74.8

Add 5 ul to each rxn
Pipette mix Library Prep Mix A 15X at ~130 ul and place on ice
Add 20 ul Lib Prep Mix A to each sample (viscous)
Pipette Mix 10X at 40 ul, brief centrifuge to collect
Incubate 20 C for 20 minutes (no heated lid)
Prepare at least 400 ul 85% EtOH per sample
Resuspend Illumina Purification Beads by vortex < 30s
Remove ligation rxn from incubation
Add 60 ul Illumina Purification Beads to each sample, pipette mix
Incubate 5 minutes at RT
Place on magnet 5 minutes
Remove ~135 ul supernatant
Add 200 ul 85% EtOH, incubate 30 s, and discard
Add 200 ul 85% EtOH, incubate 30 s, and discard
Pipette again to remove all EtOH
Air Dry for 2-5 minutes
Remove from magnet and add 34 ul ultra pure water, Pipette mix >10X at 32.5 ul
Incubate 5 minutes at RT
Return tubes to magnet for 2 minutes
Transfer 32.5 ul to new 0.2 ml tubes, store on ice
Thaw 4X PCR MM and UDI Index Mix strip
Add 5 ul Unique UDI Library Index Mix to each tube and record
Add 12.5 ul 4X PCR MM
Pipette Mix 10X at 32ul
Thermocycle with lid at 105C:

Temp

Time

Cycles

Temp

Time

Cycles

98

0:45

1

98

0:15

10

67

0:30

10

69

0:45

10

72

1:00

1

4

0:00

Hold

HOLD POINT 4 C in Thermocycler overnight

B1
B2
B3
B4
Stage Reagents
Illumina Purification Beads >20 minutes
Prepare at least 400 ul 85% EtOH per sample
Resuspend Beads by vortex
Add 45 ul to PCR rxns
Add 76 ul Illumina Purification Beads to each
Mix by pipette 15X at 160 ul
Incubate 5 minutes at RT
Place tubes on Magnet for 5 minutes
Discard ~172 ul supernatant
Add 200 ul 85% EtOH, incubate 30 s, and discard
Add 200 ul 85% EtOH, incubate 30 s, and discard
Pipette again to remove all EtOH
Air Dry for 2-5 minutes
Remove tube(s) from magnet, Add 21 ul low EDTA TE, Mix pipette 10X 20ul
Incubate 5 minutes at RT
Return tubes to magnet for 2 minutes
Transfer 20 ul supernatant to new tubes

HOLD POINT -20 C long term

B1
B2
B3
B4
Tape Station Each library
qPCR or Qubit each library