Wagner Trout 5 (5Trout)

Setup Notes

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

Cleanup each full pool via ultra purification
Size select for 300-366bp fragments size window via Pippin Prep
Mix of on campus and Sending Out for Sequencing
5% PhiX spike

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Pooling Notes

Library 1: MPR1; MPR2; YCT6

Library 2: MPR3; MPR5; GYCT3

Library 3: MPR4; MPR6; GYCT7

Quantify and normalize samples:

Normalize plate reader with TE for all plates
Quantify all plates
Normalize to 30 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 9 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	860	989
5M NaCl	0.12	860	103.2
1 mg/ml BSA	0.6	860	516
H2O	0.73	860	628
MseI (enzyme)	0.12	860	103.2
EcoR1 (enzyme)	0.28	860	241
Total	3	860	2580

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 of the 7 new plates and the plates from 1BROSE.

Reagent	ul/rxn	rxns	ul needed (1.6x)

Reagent	ul/rxn	rxns	ul needed (1.6x)
MseI oligo	1	1400	1400
H2O	0.112	1400	156.8
10x T4 Buffer	0.1	1400	140
5M NaCl	0.01	1400	14
1 mg/ml BSA	0.05	1400	70
T4 DNA ligase	0.1675	1400	234.5
Total	1.4	1400	1960

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.

GTL Plate	EcoR1 MID plate	Pool Plate	Library	Norm to	Sequencer

GTL Plate	EcoR1 MID plate	Library	Norm to	Sequencer
MPR1	1	1	30	Next
MPR2	2	1	30	Next
YCT6	3	1	30	Next
MPR3	1	2	30	Next
MPR5	2	2	30	Next
GYTC3	7 or 8	2	30	Next
MPR4	1	3	30	Next
MPR6	2	3	30	Next
GYTC7	8 or 7	3	30	Next

Label reaction plates with MID plate used.

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

Add 68 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	350	3094	4760
5x iProof buffer	4	350	1300	2000
10 mM dNTPs	0.4	350	130	200
50 mM MgCl2	0.4	350	130	200
5 uM Illumina Primers	1.33	350	432	665
iProof TAQ	0.2	350	65	100
DMSO	0.15	350	49	75
total	16	350	5200	8000

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:

Temp C	Cycles	Time

Temp C	Cycles	Time
95*	1X	3:00
98	30X	0:30
60	30X	0:30
72	30X	0:40
72	1X	10:00
4*	1X*	0:00*

*pause step

Extra PCR

Add 2.155 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed (x 1.6)
5x Iproof buffer	0.425	360	230
10 mM dNTPs	0.4	360	92
Primers	1.33	360	306
Total	2.155	360	496

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

Temp C	Cycles	Time

Temp C	Cycles	Time
98	1X	3:00
60	1X	2:00
72	1X	10:00
4	1X	0:00

Pool Using PCR Plates with Assist Plus

Organize plates by Library # above, vortex, and quick spin

Use 4GbsPoolx8

Use single channel to combine 48 ul from each well of a column into a separate labeled tube.

Pippin Prep Size Select (300-366 bp select):

First attempt showed too little product. I will concentrate via speedvac and cleanup concentration via Nucleofast 96 PCR.

Run Final Products on Tapestation for size check

Run Final Product on qPCR for check and quant

Result from qPCR check :

Sample Name	qPCR	Sample ul	RSB ul

Sample Name	qPCR	Sample ul	RSB ul
Exp5Trout1	1.86 nM	53.8	46.2
5Trout1Cleaned	9.02 nM	11.1	88.9
5Trout1Org	3.05 nM	32.8	67.2

load =800 pM

80 ul 1 nM stock, 4 ul 1 nM PhiX, 16 ul RSB