Yeast Cells Fluo

Nextera DNA Sample Preparation

 

Using :

Nextera DNA Sample Prep Kit (Illumina : ref 15028212 for 24 samples) Stored at -20°C

Nextera Index Kit (Illumnia : ref : 15028220 for 24 indices- 96 samples) Stored at -20°C

Qubit ds DNA BR Assay kit (Invitrogen : ref : Q-32850)

DNA Clean and Concentrator-5 (Proteigene ref : D4013 for 50 col)

Agilent HS DNA kit (Agilent : ref : 5067-4626)

Agencourt AMPure XP beads/kit (Beckman Coulter ref : A63881 for 60 mL)

 

TAGMENTATION OF GENOMIC DNA

Preparation

1- Thaw on ice Tagment DNA Buffer (TD) and Tagment DNA enzyme (TDE1).

For next step of the protocol, ensure ST Buffer is at room temperature.

2- After thawing, ensure all reagents are adequately mixed by inverting the tubes 3-5 times, followed by a brief spin in a microcentrifuge.

Ensure the reaction is assembled in the order described for optimal kit performance. The reaction does not need to be assembled on ice.

Procedure

1- Label tubes and add 20ul of genomic DNA at 2.5ng/uL (50ng total) to each tube.

To obtain an accurate quantification of the DNA library, it is recommended to quantify the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit ds DNA BR Assay system. (see Specific protocol). Nanodrop should be avoided.

2- Add 25uL of TD Buffer.

3- Add 5uL of TDE1. Gently pipette up and down 10 times to mix. Close tubes.

4- Centrifuge at 280g at 20°C for 1min.

5- Run on thermocycler (Ensure that lid is heating during incubation) with the following program :

55°C for 15 minutes

Hold at 10°C


CLEAN UP OF TAGMENTED DNA

Using : DNA Clean and Concentrator-5 (Proteigene ref : D4013 for 50 col) and Agilent HS DNA kit (Agilent : ref : 5067-4626)

Preparation

1- Thaw at room temperature the Resupension Buffer (RSB).

Procedure

1- Label tubes and add 180ul of Zymo DNA binding buffer.

2- Add 50uL of DNA tagmented. Gently pipette up and down 10 times to mix.

3- Transfer sample mixture to a Zymo-Spin Column in a collection tube.

4- Centrifuge for 30 secondes at 10000g. Discard the flow-through.

5- Add 200uL DNA Wash Buffer to the column. Centrifuge for 30 secondes at 10000g.

6- Repeat step 5 and centrifuge in addition 1 min at 10000g.

7- Transfer the column to a 1.5mL tube and add 25uL of RSB to the column matrix. Incubate at RT for 1 min.

8- Centrifuge 1 min at 10000g to elute the DNA.

Optional but recommanded : check the products of tagmentation reaction by loading 1uL of undiluted Zymo eluate on a HS Bioanalyzer chip. This should produce a broad distribution of DNA fragments with a size range from 150bp to 1.5kb. (see Agilent HS DNA protocol). During this control keep samples on ice, do not freeze them.

PCR SET UP

Preparation

1- If less than of a full set of libraries is pooled for sequencing, ensure that correct index 1 (i7) and index 2 (i5) primers have been selected. See the Dual Indexing and Low Plexity Pooling Guidelines section at the end of theNextera DNA Sample Preparation Guide.

2- Thaw at room temperature the PCR Master Mix (NPM), the PCR Primer Cocktail (PPC) and index primers (it takes approximatively 20 min).

3- After thawing, ensure all reagents are adequately mixed by inverting the tubes 3-5 times, followed by a brief spin in a microcetrifuge.

Procedure

1- On PCR tubes, add 5 uL of index 1 primers (orange caps) and 5 uL of index 2 primers (white caps) on corresponding tubes.

To avoid index cross-contamination, discard orange and white original caps and apply new caps (same colors) provided in the kit.

2- Add 15 uL of NPM to each tube containing primers.

3- Add 5 uL of PPC to each tubes containing NPM and primers.

4- Transfer 20 uL of purified tagmented DNA and gently pipette up and down 3-5 times. Close tubes.

5- Centrifuge at 280g at 20°C for 1min.

6- Run on thermocycler (Ensure that lid is heating during incubation) with the following program :

72°C for 3 min

95°C for 30 sec

5 cycles of :

                -98°C for 10 sec

                -63°C for 30 sec

                -72°C for 3 min

Hold at 10°C

SAFE STOPPING POINT : If you do not plan to immediately proceed to PCR clean-up following the completion of PCR, tubes can remain on the thermal cycler overnight, or you can store it at 2° to 8°C up to 2 days.

PCR CLEAN-UP

Using : Agencourt AMPure XP beads/kit (Beckman Coulter ref : A63881 for 60 mL)

Preparation

1- Bring the AMPure XP beads to room temperature.

2- Prepare fresh 80% ethanol from absolute ethanol (Important to always have fresh prepared one)

Procedure

1- Centrifuge at 280g at 20°C for 1min at RT to collect condensation.

2- Transfer PCR products on news PCR tubes (50 uL).

3- Vortex the AMPure XP beads for 30 sec.

4- Add 30 uL (for 150 Pair-end) or 25 uL for 2*250 run on MiSeq) of beads to each tubes. Gently pipette up and down 10 times.

5- Incubate at RT without shaking for 5 min.

6- Place the PCR tubes on a magnetic stand for 2 min (or until supernatant has cleared).

7- With tubes on the magnetic stand, remove and discard the supernatant.

If any beads are inadvertently aspirated into the tips, dispense the beads back to the tube and let the tube rest on the magnet for 2 min.

8- With tubes on the magnetic stand, wash the beads as follows :

-Add 200 uL freshly prepared 80% ethanol to each sample tube. You should not resuspend beads at this time.

-Incubate 30 sec on the magnetic stand (or until supernatant has cleared).

-Carefully remove and discard the supernatant.

9- Repeat step 8 and with fine pipette tips remove excess of ethanol.

10- With tubes on the magnetic stand, allow the beads air-dry for 15 min.

11- Remove tubes from the magnetic stand and add 32.5 uL of RSB to each tube. Gently pipette up and down 10 times.

12- Incubate at RT for 2 min.

13- Place the PCR tubes on a magnetic stand for 2 min (or until supernatant has cleared).

14- Transfer 30 uL of the supernatant to a new tube.

STOPPING POINT : You can now keep samples at -20°C.

 

 

Your Genomics platform such as ProfileXpert will then probably do the following:

VALIDATE LIBRARY :

Quantify libraries : On Qubit

Quality control : On an Agilent Technologies 2100 bioanalyzer with HS DNA chip

POOL LIBRARIES