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
Send 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 Buffer AE 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	230	115
5M NaCl	0.12	600	72	24	12
1 mg/ml BSA	0.6	600	360	120	60
H2O	0.73	600	438	146	73
MseI (enzyme)	0.12	600	72	24	12
EcoR1 (enzyme)	0.28	600	168	56	28
Total	3	600	1800	600	300

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	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
WCR31	1	1+2	1
WCR32	2	1+2	1
WCR33	3	3	1
WCR34	4	4+5	2
WCR36	5	4+5	2
WCR37	6	6+7	2
WCR38	7	6+7	3
WCR39	8	8	3

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)
H2O	9.52	350	3094	1904
5x iProof buffer	4	350	1300	800
10 mM dNTPs	0.4	350	130	80
50 mM MgCl2	0.4	350	130	80
5 uM Illumina Primers	1.33	350	432	266
iProof TAQ	0.2	350	65	40
DMSO	0.15	350	49	30
total	16	350	5200	3200

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
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	135.1
10 mM dNTPs	0.4	360	144	127.2
Primers	1.33	360	479	423
Total	2.155	360	776

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

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:

load =800 pM

dilute 1 nM PhiX to 500 pM