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Murine B-cells Illumina Prep 1

Murine B-cells Illumina Prep 1

Owned by Gregg Randolph
Last updated: Apr 02, 2025

Messed up with B1, T1, NB1, and NT1 added 10x less Resus than needed. Will add the correct amount to try and recover

  • 36 total samples to be processed in 6 batches, maybe 35?

    • Batch1: 2 sample batch

    • Batch2: 2 sample batch

    • Batch3: 16 samples

    • Batch4: 16 samples

Sample

Processing #

Batch

Index

Cell Count

Error

ul needed for 2500 c/ul

Viability %

Sample

Processing #

Batch

Index

Cell Count

Error

ul needed for 2500 c/ul

Viability %

B1

1

1

1

3.73X106

149

1492

55.5

B2

2

1

 

1.46 X106

58

584

60.7

B3

3

2

 

1.45 X106

58

580

58.7

B4

4

2

 

2.27 X106

90

908

69.2

B5

5

3

 

2.94 X106

118

1176

66.9

T1

6

3

2

1.13 X106

45

452

59.2

T2

7

3

 

4.26 X105

17

170.4

61.9

T3

8

3

 

1.09 X105

4.4

43.6

23.3

T4

9

3

 

2.79 X105

11

111.6

34.2

T5

10

3

 

3.99X105

16

159.6

66.4

TL1

11

3

 

4.04 X105

16

161.6

60.7

TL2

12

3

 

1.26 X106

50

504

61.2

TL3

13

3

 

1.54 X106

62

616

57.1

TL4

14

3

 

1.24 X106

50

496

73.5

KL1

15

3

 

6.12 X105

24

244.8

70

KL2

16

3

 

2.38 X106

95

952

79.2

KL3

17

3

 

1.36 X106

14

144

71.9

KS1

18

3

 

1.90 X107

760

7600

77.5

KS2

19

3

 

1.36 X107

544

5440

78.9

KS3

20

3

 

9.72 X106

389

3888

75.1

NB1

21

4

3

180000

72

720

48.5

NB2

22

4

 

1800000

72

720

48.5

NB3

23

4

 

1800000

72

720

48.5

NT1

24

4

4

180000

7.2

72

44.6

NT2

25

4

 

180000

7.2

72

44.6

NT3

26

4

 

180000

7.2

72

44.6

NKT1

27

4

 

1070000

42.8

428

67.6

NKT2

28

4

 

1070000

42.8

428

67.6

NKT3

29

4

 

1070000

42.8

428

67.6

NL1

30

4

 

3390000

135.6

1356

48.5

NL2

31

4

 

3390000

135.6

1356

48.5

NL3

32

4

 

3390000

135.6

1356

48.5

NS1

33

4

 

6040000

241.6

2416

80.7

NS2

34

4

 

6040000

241.6

2416

80.7

NS3

35

4

 

6040000

241.6

2416

80.7

 

36

4

 

 

 

 

 

1Cell Preparation

B1
B2
B3
B4
Thaw and stage capture and lysis materials
Thaw one PIP tube per sample and RNase Inhibitor on ice
Stage at RT Partitioning Reagent, Chemical Lysis Buffer 3
Stage Combination 1.5 ml/0.5 ml tube stand, P200 and P1000 wide and standard
Stage one 0.5 ml tube per sample

Reagent

Initial Conc

Final Conc

V per rxn

2.2

17.6

All

Reagent

Initial Conc

Final Conc

V per rxn

2.2

17.6

All

SSC (X)

20

3

180

396

3,168

7920

DTT (uM)

1000

1

1.2

2.6

21.1

52

RNAse Inhibitor (U/μL)

40

0.2

6

13.2

105.6

264

BSA (% by mass)

10

1

120

22

387.2

440

Nuclease-free water (μL)

 

 

892.8

1,964

15,713

39280

Total

 

 

 

 

 

 

Equilibrate the fixed cells on ice to 4 °C (starting from –20 °C storage).
Spin the cells at 500 x g for 5 minutes, preferably at 4 °C.
Aspirate and discard the supernatant (methanol and DSP. Be sure to dispose appropriately)

While resuspending, assume a loss of 40-50% of input cells.

Resuspend the cell pellet using chilled Resuspension Buffer to achieve the appropriate cell concentration based on the planned scale of your PIPseqTM kit.

NOTE: For 1 million cells, a final resuspension volume of ~100 μL should result in a concentration of ~5000 cells/μL which is optimal for the T20 format. In the case of lower cell concentration than desired, an additional centrifugation step can be introduced to adjust the resuspension volume.

Determine cell concentration using the laboratoryʹs preferred method (e.g. AO/PI staining on an automated cell counter). Dilute the cells to the working concentration (1,250 live cells/ul) with Resuspension Buffer, leaving everything on ice.

Capture and Lysis

Centrifuge Thawed PIP tubes for ~5 seconds and return to ice
Preheat Dry Bath with 0.5 ml block to 25 C (lid +5C or 30 C)
Use P200 wide bore to gently mix cell suspension 10x
Add 4 ul cell suspension directly into PIPs
Add 1-2 ul RNase Inhibitor directly into PIPs slowly
Mix PIP mix with standard bore low retention P200 at 25 ul (avoid bubbles) 10x
Add 280 ul Partitioning Reagent down the side of tube
Cap tubes and place in the yellow rotating vortex adapter in horizontal config
vortex 3000 rpm 15 sec
rotate adapter to vertical config and vortex 3000 rpm for 2 minutes
Place PIP tubes in 0.5 ml side of stand. Let stand 30 s
Place a P200 set at 115 ul toward bottom of tube and wait 5 s then slowly aspirate
Repeat above: P200 at bottom, 5 s, slowly aspirate
Prepare Chemical Lysis Buffer 3 per rxn
Get a 0.5 ml tube from kit per rxn
Label CLB3
Check Chemical Lysis Buffer for crystals, if present warm w dry bath, vortex 10s, centrifuge
Add 40 ul CLB 3 to each 0.5 ml tube with low retention P200
Add 120 ul Partitioning Reagent to each 0.5 ml tube
Vortex 0.5 ml tube 10s, then immediately pipette 160 ul to PIP
Mix PIPs by inversion >10x
Insert PIP tubes into dry bath and select skip and yes to run:

 

T2 PIP Cell Lysis

 

 

T2 PIP Cell Lysis

 

Preheat

25 C

0:00

Step 1

25 C

15:00

Step 2

37 C

45:00

Step 3

25 C

10:00

Step 4

20 C

0:00

 

HOLD POINT IN DRY BATH UP TO 96 HOURS

mRNA isolation

B1
B2
B3
B4
Thaw and stage materials
Thaw at RT Breaking Buffer for >20 m, DePartitioning Reagent
On ice Washing Buffer, RT Additive Mix V, one tube TSO per 2 samples
Stage and label per sample 1.5 ml Safe Locks, 1.5/0.5 ml stand, red PIPseq guide rack
Aliquot 1 ml Washing Buffer into 1.5 ml tubes and place in ice
0.2 ml 8 tube strips and lids
Remove PIPtubes and place in blue stand, allow to settle for 30s if needed
Place P200 at bottom of tube slowly, wait 5 s, aspirate ~130ul slowly, wipe tip along side slowly
Add 200 ul Breaking Buffer to each PIP along side
Add 40 ul DePartitioning Reagent to each PIP along side
Invert to break emulsion 10-20x
Centrifuge for 10s
Ensure distinct interface between pink bottom and cloudy top
if not, add 4 ul DePartitioning and repeat inversion and centrifuge
Remove ALL pink and red layers with P200
Centrifuge 10 s
Remove ALL remaining pink with a P20, place 0.5 ml tubes on ice
Use P200 to slowly transfer 180 ul PIPs to chilled Wash in 1.5 ml tubes
Briefly centrifuge 0.5 ml tubes
Repeat transfer of another 180 ul to 1.5 ml tubes
Place tubes in blue stand and vortex horizontally for 3 s
Centrifuge 1 m, turn off for gradual slow
Gently Place tubes back in rack
Aspirate slowly and discard supernatant to the 4 or L mark on stand
Wash2
Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
Wash3
Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
Wash4
Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
Move ~100 ul (ALL) into 0.2 ml tube (PCR strip) cap
Spin 5 s at 2000 xg and place in red rack
Let settle >1 minute
Reduce Volume to guide wire, then place on ice

cDNA synthesis

Prep Reagents
Thaw RT Enzyme Mix on ice
Prepare 1:1 Washing Buffer, 600 ul per sample, keep on ice
stage ultra pure water
Prepare RT Master Mix:

Reagent

Volume per run

2.2

8.8

17.6

Reagent

Volume per run

2.2

8.8

17.6

RT Additive Mix V

31.1

68.4

273.7

547.4

TSO

3.1

6.8

27.3

54.6

RT Enzyme Mix

4.8

10.6

42.2

84.5

Total

39

85.8

343.2

686.5

Mix well by pipetting, centrifuge briefly
Add 39 ul RT MM to each 0.2 ml well, mix well by pulse vortexing
Centrifuge <1 s
Thermocycle with lid at 105C:

Temp

Duration

Temp

Duration

25 C

30:00

42 C