2 6 2015 Library Creation for BS Samples

Today I made the libraries after doing the BS conversion yesterday. I used the Epigentek EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) that was received on January 16, 2015. Since I'm multiplexing these to run on a single lane I also used a Epigentek EpiNext NGS Barcode (Index) Set-12 for the barcoding portion following those instructions instead of the other kits instructions. I used the converted BS DNA from yesterday. Assuming there was no loss of DNA in the BS Conversion process that amounts to 111 ng per 10 ul sample. The library creation process was as follows.

All volumes in ul. All reactions mixtures made by adding reagents from largest volume to smallest. Done in 1 96 well plate with strip caps covering open wells when not in use.  Borrowed Ambion 96-Well plate magnet from Seeb Lab. Thermocycler used was Ernie, the 96 Well Plate Cycler.

dsDNA Conversion

Reaction Mixture

Component	Volume
BS DNA	10
5X Conversion Buffer	4
Conversion Primer	2
Nanopure Water	3
Total Volume	19

1. Made mixture, vortexed, centrifuged plate at 4300 rcf for 1 min 
2. Incubated in Thermocycler without heated lid
1. 5 min at 95 C
2. 5 min at 4 C
3. Added 1 ul Conversion Enzyme Mix
4. Incubated in Thermocycler without heated lid
1. 60 min at 37 C

Cleanup dsDNA

1. Resuspended MQ Binding Beads via vortex
2. Added 36 ul beads to samples
3. Vortex to mix
4. Centrifuged plate 4300 rcf for 1 min
5. Vortex to resuspend beads in sample (decided not to vortex/centrifuge again)
6. Incubate at 10 minutes at room temp
7. Place plate on magnet for 2 minutes to collect beads
8. Removed supernant
9. Washed sample with 200 ul 80% EtOH for 1 minute
10. Removed supernant
11. Repeated Wash once
12. After removing supernant, allowed beads to air dry for 10 minutes 
13. Removed Samples from magnet
14. Resuspended beads in 12 ul Elution buffer via pipetting
15. Incubated samples 2 minutes at room temp
16. Placed sample back on magnet for 2 minutes
17. Moved 12 ul supernants from Row A on plate to Row B
1. Note Albion magnet doesn't work well with samples less than 25 ul. To attempt to clean up the beads, I held the magnet at an angle depending on which side of the magnet the beads were on and ran the sample over the magnet until visibly clear. Still did not fully remove beads from sample. 

DNA End Repairing

Reaction Mixture

Component	Volume
dsDNA	11
10x End Repair Buffer	2
End Repair Enzyme Mix	1
Nanopure Water	6
Total Volume	12

1. Incubated in thermocycler with heated lid
1. 30 min at 20 C 

Cleanup End Repair DNA

1. Resuspended MQ Binding Beads via vortex
2. Added 36 ul beads to samples
3. Mixed via pipetting
4. Incubate at 10 minutes at room temp
5. Place plate on magnet for 2 minutes to collect beads
6. Removed supernant
7. Washed sample with 200 ul 80% EtOH for 1 minute
8. Removed supernant
9. Repeated Wash once
10. After removing supernant, allowed beads to air dry for 10 minutes 
11. Removed Samples from magnet
12. Resuspended beads in 12 ul Elution buffer via pipetting
13. Incubated samples 2 minutes at room temp
14. Placed sample back on magnet for 2 minutes
15. Moved 12 ul supernants from Row B on plate to Row C
1. Note Albion magnet doesn't work well with samples less than 25 ul. To attempt to clean up the beads, I held the magnet at an angle depending on which side of the magnet the beads were on and ran the sample over the magnet until visibly clear. Still did not fully remove beads from sample.

DNA dA-Tailing

Reaction Mixture

Component	Volume
End repaired DNA	12
10x dA-tailing buffer	1.5
Klenow Fragment	1
Nanopure Water	0.5
Total Volume	15

1. Incubated in thermocycler with heated lid
1. 30 min at 37 C
2. 10 min at 75 C

Adapter Ligation

Reaction Mixture

Component	Volume
dA-Tailed DNA	15
2x Ligation Buffer	17
T4 DNA Ligase	1
Adaptors	1
Total Volume	34

Note this reaction mixture is made directly in the same well as the last reaction

1. Incubated in thermocycler without heated lid
1. 10 min at 25 C

Clean Ligated DNA

1. Resuspended MQ Binding Beads via vortex
2. Added 34 ul beads to samples
3. Mixed via pipetting
4. Incubate at 5 minutes at room temp
5. Place plate on magnet for 2 minutes to collect beads
6. Removed supernant
7. Washed sample with 200 ul 80% EtOH for 1 minute
8. Removed supernant
9. Repeated Wash twice
10. After removing supernant, allowed beads to air dry for 10 minutes 
11. Removed Samples from magnet
12. Resuspended beads in 12 ul Elution buffer via pipetting
13. Incubated samples 2 minutes at room temp
14. Placed sample back on magnet for 2 minutes
15. Moved 11 ul supernants from Row C on plate to Row D
1. Note Albion magnet doesn't work well with samples less than 25 ul. To attempt to clean up the beads, I held the magnet at an angle depending on which side of the magnet the beads were on and ran the sample over the magnet until visibly clear. Still did not fully remove beads from sample.

Library Amplification

Sample Well Number, Sample ID, Barcode Used

Well Number	Sample	Barcode
1	HL28	ATCACG
2	HL26	CGATGT
3	HL31	TTAGGC
4	HL24	TGACCA
5	NF05	ACAGTG
6	NF12	GCCAAT
7	NF07	CAGATC
8	NF15	ACTTGA
9	SN08	GATCAG
10	SN04	TAGCTT
11	SN01	GGCTAC
12	SN15	CTTGTA

In the barcode placeholder is replaced with one of the 12 barcodes depending on the sample

Reaction Mixture

Component	Volume
2x HiFi Master Mix	12.5
EpiNext Universal Primer	1
Barcode (1-12)	1
Ligated DNA	11
Total Volume	25.5

1. Ran the PCR program in the thermocycler with heated lid
1. 30 sec 98 C
2. 20 sec 98 C
3. 20 sec 55 C
4. 20 sec 72 C
5. Repeat 12 times Steps 2,3,4  
6. 2 min 72 C

Cleanup library

1. Resuspended MQ Binding Beads via vortex
2. Added 25 ul beads to samples
3. Mixed via pipetting
4. Due to bubbles had to centrifuge 4300 rcf for 1 min
5. vortexed beads into suspension
6. Incubate at 5 minutes at room temp
7. Place plate on magnet for 2 minutes to collect beads
8. Removed supernant
9. Washed sample with 200 ul 80% EtOH for 1 minute
10. Removed supernant
11. Repeated Wash twice
12. After removing supernant, allowed beads to air dry for 10 minutes 
13. Removed Samples from magnet
14. Resuspended beads in 12 ul Elution buffer via pipetting
15. Incubated samples 2 minutes at room temp
16. Placed sample back on magnet for 2 minutes
17. Moved 11 ul supernants from Row D on plate to Row E
1. Note Albion magnet doesn't work well with samples less than 25 ul. To attempt to clean up the beads, I held the magnet at an angle depending on which side of the magnet the beads were on and ran the sample over the magnet until visibly clear. Still did not fully remove beads from sample.

Was not able to collect full volume of 11 ul from sample as beads were particularly troublesome in removing at this step. Attempted to clear them out using method described above but some just did not take. Without being able to measure it, it looks like most samples are between 9-11 ul in volume. 

Samples stored in -20 C freezer in 209. Will quantify with Sam next week to verify concentrations.