RNA Detection

10. 425–600μm acid-washed glass beads.


11. Phenol–chloroform–isoamyl alcohol (25:24:1) pH 6.7.


12. Chloroform.


13. 100% ethanol.


14. 80% ethanol.


15. TURBO DNaseI.


16. Nuclease-free water.


17. RNA Purification kit (e.g., GeneJET, ThermoFisher
    Scientific).

Selectively RT Tagged RNAs

Add 3' adapter via U-select primer

5' UUUUU

5' UUUUUGGGIGGIGGGIGIGG

5'

5'

UUUUUGGGIGGIGGGIGIGG

AAACCCCCCCCC

5'

5'

AAAAACCCCCCCCC

Isolate total RNA

G/I-tail all RNAs

5' TTTTTGGGGGGGGG

AAAAACCCCCCCCC 5'

NNNNNN

PCR amplify via 5' and 3' adapters and purify libraries

Poly(A) selection

Synthesize 2nd strand and

add the 5' adapter

RBPPUP

UUUUU

rRNA depletion

Analyze by paired-end

high-throughput sequencing

Section 3.1

Section 3.2

Section 3.3

Section 3.4

Section 3.5

Section 3.6

Section 3.7

Fig. 1Schematic of the RNA Tagging library preparation workflow. Adapted from
Fig. 1 in Lapointe et al. [10]

RNA Tagging Library Preparation 457