HMax Hamilton RFS

Final processed data shared with Jill Hamilton on 13/09/2022

This r-script was used to transform this MISO Tracking List to this Demux Key

Sequencing Report From University of Colorado Genomics Core:

Flowcell Summary

Clusters (Raw)	Clusters(PF)	Yield (MBases)

Clusters (Raw)	Clusters(PF)	Yield (MBases)
1,276,674,048	908,405,641	100,833

Lane Summary

Lane	PF Clusters	% of the lane	% Perfect barcode	% One mismatch barcode	Yield (Mbases)	% PF Clusters	% >= Q30 bases	Mean Quality Score

Lane	PF Clusters	% of the lane	% Perfect barcode	% One mismatch barcode	Yield (Mbases)	% PF Clusters	% >= Q30 bases	Mean Quality Score
1	457,726,974	100.00	100.00	NaN	50,808	71.71	88.45	34.68
2	450,678,667	100.00	100.00	NaN	50,025	70.6	88.08	34.61

Check In Samples Against List from Jill Hamilton

Samples Arrived 06/09/2021

Load Submission Data into MISO

Adjust Plate 1 D9 to below 150 ng/ul from 189.3 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 5 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	480	828
5M NaCl	0.12	480	86.4
1 mg/ml BSA	0.6	480	432
H2O	0.73	480	525.6
MseI (enzyme)	0.12	480	86.4
EcoR1 (enzyme)	0.28	480	201.6
Total	3	480	2160

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	480	768
H2O	0.112	480	86.016
10x T4 Buffer	0.1	480	76.8
5M NaCl	0.01	480	7.68
1 mg/ml BSA	0.05	480	38.4
T4 DNA ligase	0.1675	480	128.64
Total	1.4	480	1075.2

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

Template	EcoR1 MID plate

Template	EcoR1 MID plate
HMax_1	EcoR1 MID plate 1
HMax_2	EcoR1 MID plate 2
HMax_3	EcoR1 MID plate 3
HMax_4	EcoR1 MID plate 4
HMax_5	EcoR1 MID plate 5

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.

Pool	Plate1	Plate2	Plate3	Plate4

Pool	Plate1	Plate2	Plate3	Plate4
A	HMax_1	HMax_2	HMax_3	HMax_4

Pool	Plate1	Plate2	Plate3	Plate4

Pool

Plate1

Plate2

Plate3

Plate4

B

HMax_1

HMax_2

(FLIPPED)

HMax_3

HMax_4

(FLIPPED)

Pool	Plate5 Col 1-3	Plate5 Col 4-6	Plate5 Col 7-9	Plate5 Col 10-12

Pool	Plate5 Col 1-3	Plate5 Col 4-6	Plate5 Col 7-9	Plate5 Col 10-12
C1	HMax_5	HMax_5	HMax_5	HMax_5

Pool	Plate5 Col 1-3	Plate5 Col 6-4	Plate5 Col 7-9	Plate5 Col 12-10

Pool	Plate5 Col 1-3	Plate5 Col 6-4	Plate5 Col 7-9	Plate5 Col 12-10
C2	HMax_5	HMax_5	HMax_5	HMax_5

PCR1:

Make MM1 in a 15 ml tube:

Reagent	ul/rxn	rxns	ul needed

Reagent	ul/rxn	rxns	ul needed
H2O	9.52	240	3198.72
5x iProof buffer	4	240	1344
10 mM dNTPs	0.4	240	134.4
50 mM MgCl2	0.4	240	134.4
5 uM Illumina Primers	1.33	240	446.88
iProof TAQ	0.2	240	67.2
DMSO	0.15	240	50.4
total	16	240	5376

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 2.125 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	240	163.2
10 mM dNTPs	0.4	240	153.6
Primers	1.33	240	510.72
Total	2.155	240	827.52

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

Pippin Prep Size Select:

Pool 2 ul from all wells of the 3 final plates into an 8 tube strip. Pool 30 ul from each of these into one tube after mixing via pipette.

Run Final Product on qPCR for check

Result from qPCR check:

HMax_PP_Final: 8.05 nanoMolar

The full result report can be viewed below: