TITLE: selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing
AUTHORS: Jason D Limberis, Joel Ernst, John Metcalfe, Alina Nalyvayko

[DESCRIPTION]
Non-polyadenylated RNA includes a large subset of crucial regulators of RNA expression and constitutes a substantial portion of the transcriptome, playing essential roles in gene regulation. For example, enhancer RNAs are long non-coding RNAs that perform enhancer-like functions, are bi-directionally transcribed, and usually lack polyA tails. This paper presents a novel method, selSeq, that selectively removes mRNA and pre-mRNA from samples to enable the selective sequencing of crucial regulatory elements, including non-polyadenylated RNAssuch as long non-coding RNA, enhancer RNA, and non-canonical mRNA.

[BEFORE_START]
Prewarm SuperScript III 10X Buffer to Room temperature

[STEPS]
SECTION: poly-A tailed cDNA synthesis
1. Mix the following in a 0.2ml tube
     COMPONENT      Volume        Total   RNA      1        Oligo   dTs      1.5        10 mM   dNTP      1.5        Nuclease-free   H2O      10   

SECTION: poly-A tailed cDNA synthesis
3. Spin tube briefly and add the following and mix by pipetting
     COMPONENT      VOLUME        10X   Buffer      2        25mM   MgCl2      4        0.1   M DTT      2        Superscript  III      2   

Incubate 50 °C for 50 min followed by 5 min at 85 °C to deactivate the enzyme, then cool to 4 °C and proceed to the next step

SECTION: poly-A tailed (and ribosomal) RNA depletion
5. Add 2 µL of RNase H
SECTION: poly-A tailed (and ribosomal) DNA depletion
8. Incubate at 37 °C for 30 min
SECTION: Optional: One-step RT-qPCR quantification
17. 
     Component      10ul      12X        Luna   one step 2X      5      60        Enz   mix      0.5      6        Probemix   18s (20X)      0.5      6        GAPDH      0.5      6        RNA      2      –        H20      1.5      18   

SECTION: Optional: One-step RT-qPCR quantification
18. 
 

 

SECTION: poly-A tailed cDNA synthesis
2. Denature sample RNA/primer mixture for 5 min at 65 °C then cool to 4 °C for ≥2 min
SECTION: Optional: rRNA depletion
4. Add in the appropriate rRNA depletion oligos for you sample
Incubate 90 °C for 2 min and ramp down to 85 Room temperature at 0.1 °C per second then proceed to the next step
SECTION: poly-A tailed (and ribosomal) RNA depletion
6. Incubate 37 °C for 20 min followed by 5 min at 65 °C to deactivate the enzyme, then cool it to 4 °C and proceed to the next step
SECTION: poly-A tailed (and ribosomal) DNA depletion
7. Add in the following components and mix gently by pipetting

     Component      Volume        10X buffer      4        DNase      4        H20      10   

SECTION: Bead cleanup
9. Add 90 μl (1.8X) of resuspended RNAClean XP Beads to the sample
Mix by pipetting 10x
SECTION: Bead cleanup
10. Incubate 15 min at on ice
SECTION: Bead cleanup
11. Place on the magnet, allow the beads to aggregate, and remove and discard the supernatant
SECTION: Bead cleanup
12. Add 200 µL 80 % (v/v) ethanol and incubate (still on the magnet) for 30 s
SECTION: Bead cleanup
12.1. Remove the supernatant
SECTION: Bead cleanup
12.2. Repeat   for a total of 2 washes

SECTION: Bead cleanup
13. Air dry for30 s, don't allow the beads to become cracked
SECTION: Bead cleanup
14. Remove the tubes from the magnetic rack
Add 50 µL H20 (optionally add-in 1 µL RNase inhibitor) and resuspend the beads by pipetting ≥10x
SECTION: Bead cleanup
15. Incubate 5 min at 65 Room temperature
SECTION: Bead cleanup
16. Place on the magnet, aspirate  50 µL of the eluant into a new tube