Murine B-cells Illumina Prep 1
- Gregg Randolph
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 |
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 |