Murine B-cells Illumina Prep 1
36 total samples to be processed in 6 batches
Batch1: 2 sample batch
Batch2: 2 sample batch
Batch3: 16 samples
Batch4: 16 samples
Sample | Processing number | Batch | Index |
---|---|---|---|
| 1 | 1 |
|
| 2 | 1 |
|
| 3 | 2 |
|
| 4 | 2 |
|
| 5 | 3 |
|
| 6 | 3 |
|
| 7 | 3 |
|
| 8 | 3 |
|
| 9 | 3 |
|
| 10 | 3 |
|
| 11 | 3 |
|
| 12 | 3 |
|
| 13 | 3 |
|
| 14 | 3 |
|
| 15 | 3 |
|
| 16 | 3 |
|
| 17 | 3 |
|
| 18 | 3 |
|
| 19 | 3 |
|
| 20 | 3 |
|
| 21 | 4 |
|
| 22 | 4 |
|
| 23 | 4 |
|
| 24 | 4 |
|
| 25 | 4 |
|
| 26 | 4 |
|
| 27 | 4 |
|
| 28 | 4 |
|
| 29 | 4 |
|
| 30 | 4 |
|
| 31 | 4 |
|
| 32 | 4 |
|
| 33 | 4 |
|
| 34 | 4 |
|
| 35 | 4 |
|
| 36 | 4 |
|
1Cell Preparation
B1 | B2 | B3 | B4 |
Reagent | Initial Concentration | Final Concentration | V per rxn | 2.2 | 17.6 |
---|---|---|---|---|---|
SSC (X) | 20 | 3 | 180 | 396 | 3,168 |
DTT (uM) | 1000 | 1 | 1.2 | 2.6 | 21.1 |
RNAse Inhibitor (U/μL) | 40 | 0.2 | 6 | 13.2 | 105.6 |
BSA (% by mass) | 10 | 1 | 120 | 22 | 387.2 |
Nuclease-free water (μL) |
|
| 892.8 | 1,964 | 15,713 |
While resuspending, assume a loss of 40-50% of input cells.
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.
Capture and 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
cDNA synthesis
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 |
Temp | Duration |
---|---|
25 C | 30:00 |
42 C | 90:00 |
85 C | 10:00 |
4 C | 0:00 |
HOLD POINT 4 C in Thermocycler overnight
cDNA Amplification
Reagent | V per run | 2.2x | 8.8x | 17.6 |
---|---|---|---|---|
4X PCR MM | 15 | 33 | 132 | 264 |
1X WTA Primer | 6 | 13.2 | 52.8 | 105.6 |
Total | 21 | 46.2 | 184.8 | 369.6 |
Temp | Time | Cycles (60 ul) |
---|---|---|
95 | 3:00 | 1 |
98 | 0:15 | 5 |
69 | 10:00 | 5 |
72 | 5:00 | 1 |
4 | 0:00 | 1 |
HOLD POINT 4 C in Thermocycler overnight
Isolate cDNA from PIPs in Post PCR area
MagBead Purification
cDNA QC of remaining 2 ul
Reagent | V per rxn | 2.2X | 8.8X | 17.6 |
---|---|---|---|---|
4X PCR MM | 6.25 | 13.8 | 55 | 110 |
WTA Primer | 1 | 2.2 | 8.8 | 17.6 |
0.5X Washing Buffer | 7.75 | 17 | 68.2 | 136.4 |
Total | 15 | 33 | 132 | 264 |
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
Library Prep
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 |
Temp | Time. (50ul) |
---|---|
4 | 0:00 |
30 | 6:00 |
65 | 30:00 |
4 | 0:00 |
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 |
Temp | Time | Cycles |
---|---|---|
98 | 0:45 | 1 |
98 | 0:15 | 16 |
67 | 0:30 | 16 |
69 | 0:45 | 16 |
72 | 1:00 | 1 |
4 | 0:00 | Hold |