RNA Detection

3 Methods

The following is a detailed description of the PARIS method

(Fig.1). Indicators of successful implementation are included for

the critical steps. Standard experimental procedures like TRIzol

extraction and ethanol precipitation of RNA are not described in

details here and readers should refer to manufacturers’ manuals.

Live cells – AMT Live cells + AMT

Control lysate Crosslinked lysate

Control RNA Crosslinked RNA

Crosslinked RNA

fragments

Control RNA

fragments

Control RNA

gel slices

Crosslinked RNA

gel slices

Blank in upper

diagonal

Crosslinked RNA in

upper diagonal

Crosslinked and

ligated RNA

Ligated RNA

PARIS libraries

S1 nuclease is essential for recovery of crosslinked

RNA and fragmentation. Proteinase K removes

proteins and ensures that all crosslinking is directly

between RNA molecules

Further fragment long double stranded RNA

Duplex RNA run in base-paried conformation

Duplex RNA run in extended conformation

Only crosslinked RNA duplexes are purified

All sequenced fragments from crosslinked RNA

In vivo crosslinking of duplexes

PARIS

UV 365nm on ice and

Urea/SDS lysis

S1/PK then Trizol

purification

ShortCut digestion

First dimension

native PAGE

Second dimension

denatured PAGE

Explanation

Proximity ligation

UV 254nm reverse crosslinking

Use 20ug input RNA

Get about 10-15 ug fragmented RNA

UV 254nm reversal causes RNA damage, so the

amount of usable RNA is lower than expected.

Proximity ligation joins the ends of each duplex

Ligate pre-adenylated adapters

Barcoded reverse transcription

cDNA circularization

Library PCR and gel purification

Fig. 1Outline of the PARIS experimental strategy. Major steps are explained on theright side

66 Zhipeng Lu et al.