Wagner Trout 4 (4Trout)

Setup Notes

~28 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

Quantify and normalize samples:

Normalize plate reader with TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 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	600	690
5M NaCl	0.12	600	72
1 mg/ml BSA	0.6	600	360
H2O	0.73	600	438
MseI (enzyme)	0.12	600	72
EcoR1 (enzyme)	0.28	600	168
Total	3	600	1800

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)

Reagent	ul/rxn	rxns	ul needed (1.6x)
MseI oligo	1	600	600	400
H2O	0.112	600	67.2	44.8
10x T4 Buffer	0.1	600	60	40
5M NaCl	0.01	600	6	4
1 mg/ml BSA	0.05	600	30	20
T4 DNA ligase	0.1675	600	100.5	67
Total	1.4	600	840	560

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	Pool Plate	Library	Norm to	Sequencer
WCR31	1	1+2	1	10	Next
WCR32	2	1+2	1	10	Next
WCR33	3	3	1	10	Next
WCR34	4	4+5	2	30	Next
WCR36	5	4+5	2	30	Next
WCR37	6	6+7	2	30	Next
WCR38	7	6+7	3	30	Next
WCR39	8	8+9	3	30	Next
WCR40	1	8+9	3	30	Next
WCR41	2	10+11	4	10	Next
WCR45	3	10+11	4	10	Next
WCR46	4	12	4	10	Next
WCR47	5	13+14	6	30	Nov
WCR48	6	13+14	6	30	Nov
WCR49	7	15+16	5	40	Nov
WCR50	8	15+16	5	40	Nov
WCR51	1	17+18	5	40	Nov
WCR52	2	19+20	6	30	Nov
WCR53	3	17+18	5	40	Nov
WCR54	4	19+20	6	30	Nov
WCR43	5	21+22	7	30	Nov
WCR44	6	21+22	7	30	Nov
WCR55	7	23+24	7	30	Nov
WCR56	8	23+24	7	30	Nov
WCR57	1	25+26	8	30	Next
WCR58	2	25+26	8	30	Next
WCR59	3	27+28	8	30	Next
WCR28	7	27+28	9	10*	Next
WCR35	8	29+30	9	10*	Next
WCR42	1	29+30	9	10*	Next

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

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	153	106	212.5
10 mM dNTPs	0.4	360	144	100	200
Primers	1.33	360	479	332	665
Total	2.155	360	776	539

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):

Run Final Product on qPCR for check and quant

Result from qPCR check :

qPCR	Sample	RSB

qPCR	Sample	RSB
2.87

load =800 pM

dilute 1 nM PhiX to 500 pM

Genome Technologies Laboratory