Crosslinking and Immunoprecipitation
Followed by Sequencing (CLIP-seq)
from XRNAX Extracts

Current CLIP-seq variations typically use immunoprecipitation (IP) from total lysates for the cleanup of RNA fragments UV-crosslinked to a protein of interest [for review see Lee et al., Mol Cell, 2018]. Prefixing XRNAX to a CLIP-seq experiment comes with a number of critical advantages: 1) XRNAX extracts only contain protein and RNA. That means cellular debris, detergents, DNA or salts cannot interfere with the IP. This is especially important for proteins participating in chromatin complexes, which are not easily accessible in conventional lysates. 2.) XRNAX extracts are highly concentrated. That means much higher antibody concentrations can be achieved, or vice versa much less antibody than usually is required. 3.) As a result of the two earlier points XRNAX extracts can be fragmented using sonication instead of RNase treatment. RNase treatment usually requires laborious optimization and can lead to biases [Haberman et al., Genome Biol, 2017].

Learn here about a CLIP-seq variation starting from XRNAX extracts (refer to The Protocol section for initial XRNAX extraction).

XRNAX
Depending on the abundance of your protein-of-interest use 100-1000 µg of XRNAX extract as input material (2 confluent 245 x 245 mm2 cm dishes MCF7 cells yield approximately 1000 µg XRNAX extract).

Downstream Protocol for Crosslinking and Immunoprecipitation Followed by Sequencing (CLIP-seq) from XRNAX Extracts
The following protocol is an adaptation of the eCLIP protocol [Nostrand et al., Nature Methods, 2016] using XRNAX as an initial cleanup and the NextFlex Small RNA 3.0 kit for library preparation. The scheme further up on this page shows the workflow, by which an IP is compared to a size-matched input control (SMI-control).

IP validation
For the identification of RNA transcripts binding to a protein-of-interest validate your antibody for regular IP using the IP buffer used in the following protocol (see required material). We usually use polyclonal antibodies and validate them using MS. Make sure contaminating proteins do not have molecular weights similar to your IP target protein so they will be separated by SDS-PAGE.

Required Material
IP buffer 2 x (Tris-Cl 100 mM pH=7.5, 1 % NP40, 300 mM LiCl, 0.2 % LiDS)
Tris-Cl pH=7.5 1 M
EDTA 0.5 M
Covaris sonication tubes (Covaris, 520045)
IP-validated antibody against protein-of-interest (1-10 µg)
Protein G Magnetic Beads (Pierce, 88847)
TBST (Tris-Cl 50 mM pH=7.5, NaCl 150 mM, Tween20 0.1 %)
FastAP (Thermo, EF0651)
FastAP buffer 10 x (Thermo)
PNK (Thermo, EK0032 )
PNK buffer A 10 x (Thermo)
ATP 10 mM
RNASin Plus RNase inhibitor (Promega, N2615)
SDS-loading dye 5 x (tris-Cl pH=6.8 250 mM, SDS 10 %, 0.02 % bromphenol blue, glycerol 30 %)
DTT 1 M
4-12 % NuPAGE BisTris gel, 1 mM, 10 well (Invitrogen)
NuPAGE MES SDS Running Buffer (Invitrogen)
Nitrocellulose membrane for blotting (e.g. Biorad, Transferblot Turbo 2 µM nitrocellulose, 1704158)
Sterile scalpel
Proteinase K (Thermo, EO0491)
Proteinase K buffer (Tris-Cl 50 mM, EDTA 10 mM, NaCl 150 mM, SDS 1 %)
PCI for RNA (Roth, X985.2)
GlycoBlue (Ambion, AM9515)
NaCl 5 M
Ethanol 80 %
NextFlex Small RNA 3.0 kit (Bioo Scientific, NOVA-5132-05) or any small RNA library prepration protocol preferably using UMIs

Sonication
As an initial step RNA in XRNAX extracts is fragmented through sonication. Therefore add 1 µl tris-Cl pH=7.5  1 M along with 1 µl EDTA 0.5 M and transfer to a Covaris sonication tube. Top off with MilliQ water to fill the tube completely leaving no bubble behind (approx. 140 µl total volume). Sonicate with the following settings on a Covaris sonicator: 900 seconds, peak power 175, duty factor 50, cycles/burst 200 and average power 87.5. To produce more than 100 µg sonicated XRNAX extract per sample sonication tubes can be reused. The sonicated XRNAX extract will be used for the IP but also for the SMI-control. Both will be run alongside on the same SDS-PAGE.

SMI-Control
We start with preparing the SMI-control by reparing the RNA fragment ends to make them compatible with small RNA library preparation. For the SMI-control use 5 µg of the sonicated extract (approx. 2 µl) and mix with 33 µl MilliQ water, 5 µl FastAP buffer 10 x and 10 µl FastAP. Incubate for 15 minutes at 37 °C, then inactivate FastAP for 5 minutes at 80 °C and transfer to ice. Add 5 µl PNK buffer 10 x, 10 µl ATP 10 mM, 25 µl MilliQ and 10 PNK, mix and incubate 15 minutes at 37 °C. Combine 15 µl of the SMI-control (approx. 200 ng RNA) with 5 µl SDS-loading dye 5 x and 5 µl DTT 1 M. Heat to 70 °C for 15 minutes before running on SDS-PAGE.

IP
For the IP from approx. 900 µg XRNAX extract transfer 1000 µl sonicated XRNAX extract to a fresh 2 ml tube and combine with 1000 µl IP buffer 2 x. Add 10 µg antibody and allow binding for 4 hours at 4 °C on a rotating wheel. Wash 100 µl protein G beads three times with 1 ml IP buffer 1 x and resuspend them in 100 µl IP buffer 1 x. Add the beads to the IP and allow capture over night at 4 °C on a rotating wheel.

Capture the beads on a magnetic stand and transfer the supernatant to a fresh tube to be stored as a control. Wash the beads three times with 1 ml IP buffer, each time carefully turning the tube upside down until the beads are completely resuspended. To remove residual IP buffer wash the beads twice with 1 ml TBST while attached to the magnet.

For end repair resuspend the beads in 100 µl dephosphorylation mix (80 µl MilliQ, 10 µl FastAP buffer 10 x, 8 µl FastAP, 2 µl RNASin) and incubate 15 minutes at 37 °C, 1000 rpm shaking. Collect the beads on the magnetic stand, discard the supernatnat and wash the beads twice with 1 ml TBST. Subsequently, resuspend the beads in 100 µl PNK mix (70 µl MilliQ water, 10 µl ATP 10 mM, 10 µl PNK buffer A 10 x, 8 µl PNK, 2 µl RNASin) and incubate for another 15 mintues at 37 °C, 1000 rpm shaking. Collect the beads on the magnet and discard the supernatant.

Elute into 5 µl SDS loading dye 5 x, 5 µl DTT 1 M and 15 µl MilliQ for 15 minutes at 70 °C, collect the beads on a magnetic rack and transfer the supernatant to a fresh tube.

SDS-PAGE
Load 20 µl of the IP eluate (the rest can be kept as a control for western blotting or MS) alongside 20 µl of the SMI-control and a protein ladder on either sides onto a 4-12 % NuPAGE BisTris gel. For better excision leave out at least one well between the two samples. Run for 70-90 minutes (depending on size of target protein) using MES running buffer and subsequently blot onto nitrocellulose for 1 hour at 500 mA in the coldroom.

Proteinase K digestion & cleanup
Excise the area of the nitrocellulose membrane, which corresponds to the molecular weight of your protein-of-interest plus 75 kDA above using a clean scalpel. Cut the membrane area into pieces and transfer them to a fresh 2 ml tube. Add 200 µl proteinase K buffer and 50 µl proteinase K. Digest for 30 mintues at 55 °C. Add 250 µl PCI for RNA and mix well before 10 minutes of incubation on ice, then spin down 10 minutes with 12000 g at 4°C. Transfer 200 µl of the supernatant to a fresh tube, add 1 µl glycoblue, 12 µl NaCl 5 M, and 200 µl isopropanol. Mix and allow precipitation to occur for at least 2 hours at -20 °C. Spin down with full speed at -10 °C (or coldest setting) for 1 hour. Discard the supernatant, wash the pellet with 500 µl of 80 % ethanol, remove all residual ethanol and resuspend the pellet in 14 µl nuclease-free water.

Library preparation
RNA produced by this protocol is approx. 30-80 nt in size, carries a 5’ phosphate and a 3’ hydroxyl. For generation of sequencing libraries we use the NextFlex Small RNA 3.0 kit and gel-based size-selection of RNA fragments from 30-50 nt. This kit uses unique molecluar identifiers (UMIs), which allow for the informed removal of PCR duplicates. Note that the SMI-control is best used with UMIs, especially with small target transcripts, which upon normal deduplication can retain only very few unique reads.