Exoseq

ExoSeq and RNAseH-seq are methods for 5' and 3' sequencing, respectively. Application of this protocol to the impact of temperature on the circadian cycle network in fly heads we published in Afik et al, Nucleic Acid Research, in press

Exo-seq (5' end sequencing)

A) PolyA selection (Dynabeads Oligo(dT)25, ThermoFisher Scientific)

according to the manufactures protocol. Can start from either a cell lysat, or total RNA. 100ng-1µg input can be used.

Elute in 18µl cold water (DEPC treated).

B) Zn-based Fragmentation

a. Add 2µl of 10X Fragmentation Buffer (Fragmentation Reagents, Ambion, AM8740)

b. Incubate at 70°C for 1min 50sec

c. Add 2µl of Stop Solution and place on ice

C) 2.5X SPRI CleanUp

a. Add 55µl SPRI beads (Agencourt AMPureXP, BECKMAN COULTER)

b. Incubate for 15-20min, and then place on a magnet for 5 min.

c. Remove supernatant and wash twice with 70% EtOH keeping on the magnet (don’t disturb beads. For each wash keep beads in EtOH for about 30 sec).

d. Dry beads for 2-5 minutes (at RT, kept on the magnet).

e. Elute in 39µl of water.

D) PNK treatment

a. Heat the 39µl RNA, 70°C 2min

b. Add 5µl of PNK buffer (NEB, M0201)

c. Add 0.5µl of 100mM ATP

d. Add 1µl of RNAse inhibitor (NEB, M0314)

e. Add 4.5µl of T4 PNK (NEB, M0201)

f. Incubate for 30 min at 37°C

E) 2.5X SPRI CleanUp

a. Add 130µl SPRI beads

b. Incubate for 15-20min, then place on a magnet for 5 min.

c. Remove sup and wash twice with 70% EtOH keeping on the magnet (for wash keep beads in ethanol for about 30 sec)

d. Dry beads for 2-5 minutes

e. Elute in 24µl of water

F) 5’Phoshate dependent Exonuclease treatment (Terminator™ 5'-Phosphate-Dependent Exonuclease, Epicentre Biothechnologies, TER51020)

a. Heat to denaturate the eluted RNA at 70°C for 2min, and place immediately on ice.

b. Add 3µl of 10X Buffer A (Gently mix and briefly centrifuge the Terminator 10X Reaction Buffer A prior to use)

c. Add 1µl of RNAse inhibitor and 2µl of Terminator Exonuclease. Mix well.

d. Incubate the reaction at 30°C for 70 minutes in a thermocycler (with heated lid)

G) 2.5X SPRI CleanUp

a. Add 75µl SPRI beads

b. Incubate for 15-20min, and then place on a magnet for 5 min.

c. Remove sup and wash twice with 70% EtOH keeping on the magnet (for wash keep beads in ethanol for about 30 sec)

d. Dry beads for 2-5 minutes

e. Elute in 31µl of water.

H) FastAP +PNK treatment

a. Heat to denature RNA at 70°C for 2 minutes and place immediately on ice

b. Add:

• 10μl of x5 FNK buffer*

• 1 μl RNAse inhibitor

• 5 μl FasAP enzyme (ThermoFisher Scientific, EF0651)

• 3 μl T4 PNK

Mix well, incubate at 37°C for 20min

(* x5 FNK buffer: 250mM Tris7.5, 25mM MgCl2, 50mM DTT, 250mM KCl, 0.05% TritonX100)

I) 2.5X SPRI CleanUp

a. Add 125µl SPRI beads

b. Incubate for 15-20min, and then place on a magnet for 5 min.

c. Remove sup and wash twice with 70% EtOH keeping on the magnet (for wash keep beads in ethanol for about 30 sec)

d. Dry beads for 2-5 minutes,

e. Elute in 6µl of water.

Summary and information about intermediate products

RNAseH-seq (3' end sequencing)

A) Mg-based Fragmentation (NEBNext® Magnesium RNA Fragmentation Module, NEB, E6150S)

Start with RNA in 18μl DEPC water.

a. Add 2μl of fragmentation buffer

b. Incubate at 94°C for 2 minutes and place immediately on ice.

c. Add 2μl of stop solution

C) PolyA selection (Dynabeads Oligo(dT)25, ThermoFisher Scientific )

According to the manufactures protocol.

Elute in 12µl cold water (DEPC treated).

D) RNAseH treatment (PolyA tail removal)

a. Mix:

• 11.8µl RNA (fragmented + polyA selected)
• 5 μl oligo(dT) primer (at 600 ng/μl)
• 2μl 10x RNAseH buffer (NEB, M0297)

b. Heat at 85°C for 5 min to denature, then incubate at 42°C for 10 min followed by slow cooling to 32°C. (cool at a rate of 1°C per min in a thermocycler).

c. When the reaction reaches 32°C, add 0.5 μl RNase Inhibitor (NEB) and 0.7 μl RNase H (NEB, M0297), incubate it at 37°C for 1 hour.

d. Heat inactivate at 65°C for 20 min

E) Silane Cleanup (Dynabeads MyOne™ Silane, ThermoFisher Sientific, 37002D)

{Beads cleanup: Use 15 µl of Silane beads/sample, remove sup from the beads using a magnet, rinse beads with 500µl RLT buffer (QIAGEN, 79216). Resuspend washed beads in 60 µl RLT buffer/ sample.}

a. Add 60µl Silane beads (in RLT buffer) to the 20µl RNA. Mix well, and keep at RT for 1 min.

b. Add 30µl of 100% EtOH, mix well, and keep at RT for 2 min.

c. Place on a magnet for 5 minutes

d. Discard sup

e. Wash twice with 70%EtOH

f. Let air-dry at RT for 3-10 min

g. Elute in 16.5µl DEPC water (resuspend the beads in water, for 2 min, place 5 min on the magnet and Transfer 16µl of the RNA to a new tube)

F) FastAP treatment (ThermoScientific Scientific)

a. Heat to denature RNA (16µl ) at 70°C for 2 minutes and place immediately on ice

b. Add:

• 2μl 10XFastAP buffer
• 2μl FastAP enzyme

(Total 20µl)

Mix well, incubate at 37°C for 10min, 68°C for 2 minutes

G) Silane Cleanup

{Beads cleanup: Use 15 µl of Silane beads/sample, remove sup from the beads using a magnet, rinse beads with 500µl RLT buffer (QIAGEN, 79216). Resuspend washed beads in 70µl RLT buffer/ sample- Now you want to keep ALL sizes of RNA.}

a. Add 70µl Silane beads (in RLT buffer) to the 20µl RNA. Mix well, and keep at RT for 1 min.

b. Add 120µl of 100% EtOH, Mix well, and keep at RT for 2 min.

c. Place on a magnet for 5 minutes

d. Discard sup

e. Wash twice with 70%EtOH

f. Let air-dry at RT for 3-10 min

g. Elute in 16.5µl DEPC water

h. Transfer 16µl of the RNAto a new tube

i. Elute 6-µl DEPC water

{*Note: this cleaning step can be replaced by X2.5 SPRI cleanup / other}

Summary and information about intermediate products

Library preparation

A) First ligation (RNA/DNA)

3’ Linker ligation was done as published in Shishkin et al. Nat. Methods 2015 (step 3).

B) Pooling and Adaptor cleanup

Ligated RNA was cleaned with Silane beads (as described in “E” in the 3’-end RNA-seq protocol above) and eluted with 13µl of water. Or, in case of many samples and <1µg starting amount per sample, samples were pooled to a single tube and cleaned with ‘RNA clean and concentrator’ (Zymo Research, R1015) as described in Shishkin et al. Nat. Methods 2015 (step 4) and eluted in 13µl of water

C) First strand cDNA

a. Take 12.5 µl RNA

b. Add 1.5 µl of AR23 primer (30 µM stock, 5´-CCTACACGACGCTCTTCC-3´)

c. Mix well - Heat the mixture to 70°C for 2 min and immediately place on ice

d. Add (in order, on ice):

• 2µl of 10X RT buffer (AffinityScript, Agilent)
• 2µl DTT 100mM
• 0.8µl dNTP Mix (25mM)
• 0.5µl RNAse inhibitor (NEB, 40U/µl)
• 0.7µl AffinityScript RT enzyme

(Total 20 µl)

e. Place in HOT (55°C) incubator.

f. Incubate at 55°C for 45 minutes, 94°C – 1 min, 4°C – 1 min

D) RNA degradation after RT

Add 10% (2 µl) of 1M NaOH – incubate at 70°C for 12 minutes. Immediately neutralize with 4µl of 0.5M Acetic Acid

E) RT primer cleanup

{Beads cleanup: Use 10 µl of Silane beads/sample, remove sup from the beads using a magnet, rinse beads with 500µl RLT buffer (QIAGEN, 79216). Resuspend washed beads in 80 µl RLT buffer/ sample.}

a. Add 80µl Silane beads (in RLT buffer) To the 26µl RNA

b. Mix well, and keep at RT for 1 min.

c. Add 40µl of 100% EtOH

d. Mix well, and keep at RT for 2 min.

e. Place on a magnet for 5 minutes

f. Discard sup

g. Wash twice with 70%EtOH

h. Let air-dry at RT for 3-10 min

i. Keep Silane beads, add 5 µl of H2O to the beadsand mix well.

F) Second Ligation (ssDNA/ssDNA) 3’ Linker ligation on the beads

a. Heat at 75°C the 5 µl DNA (with beads) and 1µl 3Tr3 adapter (100 µM, 5'- AGATCGGAAGAGCACACGTCTG-3', Important: this is a DNA primer that Needs: 5’-P and 3’ ddC) for 2 min and place on ice.

b. Make ligation reaction mix consisting of:

• 2µl 10XLigase buffer (NEB, M0204)
• 1.9µl DMSO (100%)
• 0.2µl ATP (100mM)
• 9µl PEG 8000 (50%)
• 2µl T4 RNA Ligase (NEB, M0204)

Mix well , several times

Incubate over night at 22°C

G) Silane Linker cleanup

{Take extra 3 µl of Silane beads/sample, rinse with RLT, remove sup and resuspend in 60µl of RLT buffer/ sample}

a. Add 60µl of washed beads to the 20µl Ligated RNA

b. Mix well and incubate for 1 min at RT

c. Add Add 30µl of 100% EtOH

d. Mix well, and keep at RT for 2 min.

e. Place on a magnet for 5 minutes

f. Discard sup

g. Wash twice with 70%EtOH

h. Let air-dry at RT for 3-10 min

i. Elute in 25µl of water (incubate for 2 min at RT, place on a magnet for 5 min, and transfer 23µl to a new tube)

H) PCR Enrichment

Make a mix consisting of:

• 23µl of cDNA
• 1µl of primer 1 (2P_univ, 12.5 μM)^
• 1µl of primer 2 (2P_barcode, 12.5 μM)
• 25µl of High-fidelityPCR MasterMix (NEB, M0541)

(Total 50 µl)

^The PCR primers sequences are used as published in Shishkin et al. Nat. Methods 2015 .

Run using PCR program:

I) SPRI Library cleanup

Clean twice with 37 µl of SPRI beads/50 µl sample (-0.75X SPRI).

ELUTE in 12-15 µl of H2O. Load 1 µl on BioAnalyzer.

Summary

A scheme summarizing the various sequences added to the RNA fragment at each step of the library preparation is depicted below:

Tips for further optimization

The protocols described here are simple and modular and can be easily modified at many steps.

• For the Exo-seq, the starting material can be either PolyA+ selected RNA or any other ribosomal depleted RNA.
• The fragmentation reagent needs to be metal based, but it can be any metal-based fragmentation module. We calibrated the amount and incubation time of the Zn-based reagent of Ambion to give fragments of around 300 bases but this can be modified accordingly.
• The cleanup steps can be replaced with other RNA/DNA cleanup methods (Just notice to use size selection when cleaning after oligo/primer containing steps).
• The library preparation protocol can be easily modified with any other ligation based /stranded RNA-library preparation Protocol.
• Regarding the 5’ exonuclease activity - after fragmentation, the enzyme efficiency is reduced. One reason for that is that after RNA fragmentation there are more 5’ ends, thus the enzyme has more “substrate” starts. This should be considered if adapting the protocol for adding the exonuclease prior to fragmentation. Secondly, we noticed that when the fragments are pre-treated with PNK the 5’ exonuclease activity is more efficient and the sequencing data is “cleaner”. Thus, the PNK treatment step is an important step and should not be removed.