Wagner Trout 2 (2Trout)
Setup Notes
~32 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:
Pool ~4*96 individuals per library
Cleanup each full pool via ultra purification
Size select for 300-366bp fragments size window via BluePippin or Pippin Prep
Sequence each library on a NextSeq P3 flowcell
5% PhiX spike
Client Plate Name | GTL Plate Name | Low Volume Wells | Empty Wells | EcoR1 | Samples |
|---|---|---|---|---|---|
WRosenthal_Trout_plate9 | WRT9 |
|
| 1 | 96 |
WRosenthal_Trout_plate10 | WRT10 |
|
| 2 | 96 |
WRosenthal_Trout_plate11 | WRT11 |
|
| 3 | 96 |
WRosenthal_Trout_plate12 | WRT12 |
|
| 4 | 96 |
WRosenthal_Trout_plate13 | WRT13 |
| B8 | 5 | 95 |
WRosenthal_Trout_plate14 | WRT14 |
|
| 6 | 96 |
WRosenthal_Trout_plate15 | WRT15 |
|
| 7 | 96 |
WRosenthal_Trout_plate16 | WRT16 |
|
| 8 | 96 |
WRosenthal_Trout_plate17 | WRT17 |
| D5, F5 | 1 | 94 |
WRosenthal_Trout_plate18 | WRT18 |
|
| 2 | 96 |
WRosenthal_Trout_plate19 | WRT19 |
|
| 3 | 96 |
WRosenthal_Trout_plate20 | WRT20 |
|
| 4 | 96 |
WRosenthal_Trout_plate21 | WRT21 |
| G11, H11 | 5 | 94 |
WRosenthal_Trout_plate22 | WRT22 |
|
| 6 | 96 |
WRosenthal_Trout_plate24 | WRT24 |
| H6, G12, H12 | 7 | 93 |
MT_YCT_RangewideRADs_plate01 | WR_MYR1 |
|
| 8 | 96 |
MT_YCT_RangewideRADs_plate03 | WR_MYR3 |
|
| 1 | 96 |
WY_YCT_2021_plate04 | WR_WYCT4 |
| E12, F12 | 2 | 94 |
WY_YCT_2021_plate09 | WR_WYCT9 |
|
| 3 | 96 |
WY_YCT_2021_plate10 | WR_WYCT10 |
|
| 4 | 96 |
WY_YCT_2021_plate11 | WR_WYCT11 |
|
| 5 | 96 |
WY_YCT_2021_plate13 | WR_WYCT13 |
|
| 6 | 96 |
Wrosenthal_GY21_YCT_tray1 | WR_GYCT1 | G1, H1, C9, F9, H9, F10, A12-D12 |
| 7 | 86 |
Wrosenthal_GY21_YCT_tray3 | WR_GYCT3 | H1 |
| 8 | 95 |
Wrosenthal_GY21_YCT_tray4 | WR_GYCT4 | A10 |
| 1 | 95 |
Wrosenthal_GY21_YCT_tray6 | WR_GYCT6 |
|
| 2 | 96 |
Wrosenthal_PM_YCT_plate2 | WR_PMCT2 | H4, B8 |
| 3 | 94 |
Wrosenthal_PM_YCT_plate1 | WR_PMCT1 |
| H4, Col 5, A6-E6 | 4 | 82 |
WRosenthal_Trout_plate25 | WRT25 |
| C6-H6, H9, Col 10, 11, 12 | 5 | 65 |
MT_WCT_plate15 | WR_WCT15 |
| E8-H8, Col 9, 10, 11, 12 | 6 | 60 |
Wrosenthal_GY21_YCT_tray7 | WR_GYCT7 |
| H8, Col 9, Col 10, Col 11, A12-G12 | 7 | 63 |
WY_YCT_2021_plate14 | WR_WYCT14 |
| Col 11, 12 | 8 | 80 |
Check In Samples Against List from Will Rosenthal
Load Submission Data into MISO
Quantify and normalize samples:
Normalize plate reader with Buffer AE for all plates
Quantify all plates
Check for samples above 150 ng/ul
Transfer 20 ul from all wells of a plate needing normalization to new VWR plate
Add 20 ul TE to all wells above 150 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 32 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 3072 | 5,299.2 |
5M NaCl | 0.12 | 3072 | 553 |
1 mg/ml BSA | 0.6 | 3072 | 2764.8 |
H2O | 0.73 | 3072 | 2242.6 |
MseI (enzyme) | 0.12 | 3072 | 553 |
EcoR1 (enzyme) | 0.28 | 3072 | 1290.3 |
Total | 3 | 3072 | 12702.9 |
Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
|---|---|---|---|
MseI oligo | 1 | 3072 | 4915.2 |
H2O | 0.112 | 3072 | 550.5 |
10x T4 Buffer | 0.1 | 3072 | 491.5 |
5M NaCl | 0.01 | 3072 | 49.2 |
1 mg/ml BSA | 0.05 | 3072 | 245.8 |
T4 DNA ligase | 0.1675 | 3072 | 823.3 |
Total | 1.4 | 3072 | 7075.5 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate | Pool Plate | Library |
|---|---|---|---|
WRosenthal_Trout_plate9 | 1 | 2Trout_Pool1-4 | 1 |
WRosenthal_Trout_plate10 | 2 | 2Trout_Pool1-4 | 1 |
WRosenthal_Trout_plate11 | 3 | 2Trout_Pool5-8 | 1 |
WRosenthal_Trout_plate12 | 4 | 2Trout_Pool5-8 | 1 |
WRosenthal_Trout_plate13 | 5 | 2Trout_Pool9-12 | 2 |
WRosenthal_Trout_plate14 | 6 | 2Trout_Pool9-12 | 2 |
WRosenthal_Trout_plate15 | 7 | 2Trout_Pool13-16 | 2 |
WRosenthal_Trout_plate16 | 8 | 2Trout_Pool13-16 | 2 |
WRosenthal_Trout_plate17 | 1 | 2Trout_Pool21-24 | 4 |
WRosenthal_Trout_plate18 | 2 | 2Trout_Pool21-24 | 4 |
WRosenthal_Trout_plate19 |