Restriction fragment sequencing library

The four pools were mailed to the University of Colorado’s Genomics Core on 03/23/2021. Pools were ran on 03/30/21.

Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house

Used this r-script to bind the Masterkey to the pool and mid plate map, this plate map, and the 2017 plate map to make this demux key
QC from University of Colorado Genomics Core

Reagents and Ordering

EcoR1 (20,000 units/ml)
MseI (10,000 units/ml)
T4 DNA ligase buffer (400,000 units/ml)
DNA polymerase (BioRad iProof or KAPA HiFi)
BSA (1 mg/ml)-We have this in 20 mg/ul
1 M NaCl-We have this at 5M
DMSO
EcoR1 adaptors (We need 1 uM MIDed in plates)
Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
IIllpcr2 (10 uM working)
Illpcr1 (10 uM working)
Pool of above 2 primers (5 uM working of each)

Original SOP

Normalize templates

Use plate reader to quantify templates

Normalize to between 20 ng/ul and 150 ng/ul

2017 alfalfa: Transfer to conical pcr plates. Normalize on nimbus to 100 ng/ul in 30 ul.

Illumina Primer Pooling

Add 900 ul std TE to 5 tubes

Add 50 ul of both Illpcr1 and Illpcr2 to each tube

Seal, vortex, and spin tubes. You will need 2 tubes for this prep.

MseI oligo Annealing

Only needs to be done after first making working stock.

Add 1200 ul std TE to two 2 ml tubes.

Add 150 ul MseI1 to each tube

Add 150 ul Mse2 to each tube

Close, vortex, and spin down both tubes.

Heat to 95C for 5 minutes and allow to slowly cool to RT.

EcoRI plate 4 oligo Annealing

SpeedVac Stock Plate 7 and Stock Plate 8 to dry oligos

Resuspend with 20 ul H2O and 60 ul std TE to avoid over saturating with Tris and EDTA to create 25 uM stocks.

Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.

Label it “GBS Working Stock Plate 4” Seal, Vortex, and Spin down resulting plate.

Heat to 95C for 5 minutes and allow to slowly cool to RT

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 16 plates using the Benchsmart

Reagent	ul/rxn	rxns	ul needed
10x T4 Buffer	1.15	1800	2070
5M NaCl	0.12	1800	216
1 mg/ml BSA	0.6	1800	1080
H2O	0.73	1800	1314
MseI (enzyme)	0.12	1800	216
EcoR1 (enzyme)	0.28	1800	504
Total	3	1800	5400

Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.

Add 6 ul template to each plate with 8 channel. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with Benchsmart.

Reagent	ul/rxn	rxns	ul needed
MseI oligo	1	1800	1800
H2O	0.112	1800	201.6
10x T4 Buffer	0.1	1800	180
5M NaCl	0.01	1800	18
1 mg/ml BSA	0.05	1800	90
T4 DNA ligase	0.1675	1800	301.5
Total	1.4	1800	2591.1

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

Template	EcoR1 MID plate
2017_ALFALFA_PLATE5	5
2017_ALFALFA_PLATE6	6
2017_ALFALFA_PLATE7	7
2017_ALFALFA_PLATE8	8
2017_ALFALFA_PLATE9	1

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate at 16C for 6 hours.

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Pool ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:

Pool	Plate1	Plate2	Plate3	Plate4
1	~~2017_ALFALFA_PLATE1~~	~~2017_ALFALFA_PLATE2~~	~~2017_ALFALFA_PLATE3~~	~~2017_ALFALFA_PLATE4~~
2
3
4
5
~~6a (cycler)~~	~~2017_ALFALFA_PLATE10~~	~~2017_ALFALFA_PLATE11~~
~~6b (incubator)~~	~~2017_ALFALFA_PLATE10~~	~~2017_ALFALFA_PLATE11~~

We are running duplicate PCRs for each template.

Add 16 ul of PCR1 MM to each well of plates

Reagent	ul/rxn	rxns	ul needed
H2O	9.52	900	8568
5x iProof buffer	4	900	3600
10 mM dNTPs	0.4	900	360
50 mM MgCl2	0.4	900	360
5 uM Illumina Primers	1.33	900	1197
iProof TAQ	0.2	900	180
DMSO	0.15	900	135
total	16	900	14400

Add 4 ul of restriction/ligation products to each well

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 2.125 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	900	382.5
10 mM dNTPs	0.4	900	360
Primers	1.33	900	1197
Total	2.155	900	1939.5

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

Pooling

Sequencing Pool	PCR Pool	PCR Pool
seqPool1	Pool1	Pool2
seqPool2	Pool3	Pool4
seqPool3	Pool5	Pool6

Size Selection

We will utilize the Pippin Prep machine on loan from the Ernest Lab.