DSN Preparation

updated 2019-12-19

Supplies
item	quantity
tips_100	22.0
tips_100	5.0
tips_10	3.0
tubes_200ul	3.0
tubes_1.7ml	1.0
tips_1000	3.0
1_M_HEPES_ul	200.0
5_M_nacl_ul	400.0
ph2o_ml	0.4
dsn_kit	1.0
tube_screw_1.5ml	1.0

DSN normalization is critical ensuring an even distribution of coverage across probes. There are a genes that are highly expressed in all cells and DSN normalization helps to remove these high abundance probes and transcripts.

DSN needs to be properly diluted and should be tested for activity levels before proceeding

The protocol below was taken from Illumina’s recommendations

Reagents

Reagent	Supplier
1 M HEPES buffer solution	Invitrogen, part # 15630‐080
5 M NaCl solution	Ambion, part # AM9760G
KAPA HiFi HotStart PCR kit with dNTPs	Kapa, part #KK2502
Strip tubes	General lab supplier
DSN Kit	Evrogen, part # EA001 Sigma Aldrich, part # E7023
Ethanol 200 proof (absolute) for molecular biology (500 ml)	AB, part # 4333764F
PCR Primer PE 1.0	Included in Kapa stranded mRNA kit
PCR Primer PE 2.0	Included in Kapa stranded mRNA kit
SPRI beads	Agencourt AMPure, part # 29152; KAPA Pure Beads, part #KK8000
Nuclease-free water	General lab supplier

Equipment

• Thermocycler
• Magentic stand compatible with strip tubes

Things that can be done ahead of time

1. Pool individual RNA libraries in equal quantities to create a single pool of 500 ng. This pool will now be called the sample. For example pool 125 ng each of four individual libraries.

2. Create a 4X hybridization solution and store in a screw cap tube for future use at -15˚C to -25˚C: This recipe has been cut in half to fit the 0.5mL screw cap tubes we currently have in stock, it normally is for 1000 uL, this recipe is prepared for 1 library(ies) pooled from 22 sample(s).

Reagent	Volume_ul
1 M HEPES buffer solution	100
5 M NaCl solution	200
Nuclease free water	200
Total Volume Per sample	500

3. Set one side thermocycler or heat block to hold at 68˚C and pre-heat it.

Procedure

1. Prepare the following reaction mix in a separate, sterile, nuclease‐free 200 ul PCR tube on ice for each sample to be normalized.

Component	Volume
Sample library (500 ng)	13.5 ul
4X Hybridization buffer	4.5 ull
Total Volume Per Sample	18 ul

• NOTE: The sample library volume is optimized for the removal of rRNA from a non‐poly‐A selected library. For a reduction of highly expressed transcripts from a poly‐A selected library, more sample library may be needed for optimal results.*

2. Gently pipette the entire volume up and down 10 times, then centrifuge briefly to mix.
   

3. Transfer the entire volume of reaction mix directly to the bottom of a new, sterile, nuclease‐free 200 ul PCR tube, using a pipette. Do not let the sample contact the side of the tube during the process.

4. Incubate the reaction mix tube on the thermal cycler using the following PCR cycling conditions:

Step	Temp	Duration
Initial denaturation	98 °C	2 min
Treatment	68 °C	5 hours

Caution: Following incubation, keep the thermal cycler lid closed and the temperature held at 68°C. Do not remove the reaction mix tube from thermal cycler prior to and during DSN treatment.