Wagner Trout - First 12 plates (Trout1)

Setup Notes

~40 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

• Only 2-3 plates will be included in each pool

• Pooling of samples for PCR will either only be done in groups of three or handled by half plates in sets of 4

•  Each library should be sequenced on a 1x100bp run on a NovaSeq SP flowcell – doing this at the sequencing facility you usually use in Denver should work just fine for us. I believe you’ve previously mentioned that each run with these specifications costs approximately $3,100.

• Please size select each library for the 300-350bp fragment size window via Pippin Prep

• Multiplex ~3 plates-worth of individuals per library – the following arrangements of plates within libraries partially accounts for differences in sample age, extraction method, and hopefully concentration.

  • MT_YCT_RangewideRADs_plate2, WY_YCT_2021_plate7, WY_YCT_2021_plate12, and WRosenthal_plate8_Dec21 (if possible) should be together

  • WRosenthal_plate1_Dec21, WRosenthal_plate2_Dec2021, and WRosenthal_plate3_Dec21 should be together

  • WRosenthal_plate4_Dec21, WRosenthal_plate5_Dec2021, and WRosenthal_plate6_Dec21 should be together

  • WRosenthal_plate7_Dec21, GY21_YCT_tray2, and GY21_YCT_tray5 should be together

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 Trout5 - Trout13

• 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 11 plates using the 8 channel

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.

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). C1 and C2 will be recombining Plate 5 column wise. Combine columns 1-3, 4-6, 7-9 and 10-12 into columns 1-3 of a new plate. Then, to columns 7-9 of the same plate, add columns 1-3 and 7-9. Flip Plate 5 around and then pipette 6-4 and 12-10 into columns 7-9 of the pool plate. Mix together 20 ul from each well of each plate for pooled plate using the Benchsmart. After pooling all samples, vortex and spin down plates then use 4 ul from the pooled plates for PCR templates.

PCR1:

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

*pause step

Extra PCR

Add 2.155 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Pippin Prep Size Select (350-450 bp select):

Run Final Product on qPCR for check

Result from qPCR check: