TITLE: TLC-CLIP 
AUTHORS: Christina Ernst, Julien Duc, Didier Trono

[DESCRIPTION]
RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to increase the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss, allowing the profiling of RNA-protein interactions from as few as 1000 cells.
In the accompanying manuscript, we have applied TLC-CLIP to four endogenous RNA-binding proteins, demonstrating its reproducibility and improved precision due to a higher number of crosslinking-induced deletions that serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

[BEFORE_START]
Prepare Buffers

iCLIP Lysis Buffer
 

High Salt Wash Buffer

 


PNK Wash Buffer

 

5X PNK Buffer

 

4X Ligation Buffer

 


Proteinase K Buffer

 

Oligo(dT) Binding Buffer

 


Oligo(dT) Wash Buffer

 

First-Strand (FS) Buffer (5X)

Final ConcentrationStock SolutionFor 50 ml10 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.40.5 ml150 mM LiCl8 M LiCl0.9375 ml0.1 mM EDTA0.5 M EDTA0.01 mlAdd H2O48.5525 ml
 

Generation of preadenylated TLC-L3 adapter

TLC-L3 oligo was ordered from IDT at 250 nmole scale, carrying a 5' phosphorylation and 3' IRDye® 800CW (NHS Ester) (v3) modification and purified using RNase-free HPLC with a total yield of 21.1 nmoles. 

Set up 50 µl of 100 µM TLC-L3 adapter (5 nmoles) with 25 µl 10X 5' DNA Adenylation Reaction Buffer, 25 µl 1 mM ATP and 50 µl Mth RNA Ligase (1nmol) in a total volume of 200 µl using the . 
Incubate at 65ºC for 2 hours followed by inactivation at 85ºC for 10 minutes (reaction turns cloudy). 
Clean up using the   by mixing the 200 µl preadenylation reaction with 4.8 ml PNI buffer and distributing over 10 columns of Nucleotide Removal Kit. 
Spin down at 6000 rpm for 30 seconds.
Wash once in 750 µl PE and spin for 1 minute at 6000 rpm, followed by an empty spin at full speed. 
Transfer to a new collection tube and add 50 µl H2O per column and incubate at RT for 2 minutes
Spin at 6000 rpm for 1 minute to elute
Combine eluates at an approximate final concentration of 10 µM and prepare 1 µM working stocks to be stored at -20ºC. 

[GUIDELINES]
While working with RNA during the inital parts of the protocol, keep samples cold (on ice) and in RNase-free environment. Use RNase-free water for reactions and buffers. 

[STEPS]
SECTION: UV crosslinking and generation of cell lysates
1. UV crosslinking and generation of cell lysates

Grow desired cell line to ~80% confluency in appropriate culture conditions.
SECTION: UV crosslinking and generation of cell lysates
91. Sonicate lysates for 10-20 seconds at 0.5sec ON and 0.5sec OFF at 10% amplitude to reduce viscosity
SECTION: Preparation of bead-antibody mixture
2. 
Preparation of bead-antibody mixture
We routinely use 1-2 µg of antibody per IP in 25-50 µg of cell lysate. 
 

SECTION: Preparation of bead-antibody mixture
2.1. Wash appropriate amount of beads twice in 1 ml iCLIP Lysis Buffer, then resuspend in 100 µl per antibody
SECTION: UV crosslinking and generation of cell lysates
1.1. Remove media, wash once in ice-cold PBS and drain cells of all fluid.
SECTION: UV crosslinking and generation of cell lysates
1.2. Transfer the plate onto ice, remove lid of the culture dish, and crosslink at 254 nm with 300 mJ/cm2. 
on ice 
 

SECTION: UV crosslinking and generation of cell lysates
1.3. Scrape cells into 5 ml PBS, count then aliquot desired number of cells and spin down.
SECTION: UV crosslinking and generation of cell lysates
1.4. Resuspend cell pellet in iCLIP Lysis Buffer - 1 ml buffer for 1 million cells - scale accordingly.

SECTION: UV crosslinking and generation of cell lysates
1.5. Place lysates on ice for 5 minutes.
on ice 5 min 
SECTION: UV crosslinking and generation of cell lysates
1.6. Sonicate lysates for 10-20 seconds at 0.5sec ON and 0.5sec OFF at 10% amplitude to reduce viscosity. 

-80 °C 

SECTION: Preparation of bead-antibody mixture
2.2. Add antibody to beads and incubate at room temperature for 30-60 minutes rotating. 

Room temperature 
30 min 
 


SECTION: Preparation of bead-antibody mixture
2.3. Wash beads twice in 1 ml iCLIP Lysis buffer then resuspend in 10 µl 6X Protease Inhibitor per IP.

 

SECTION: UV crosslinking and generation of cell lysates
1.7. Quantify cell lysates with Pierce BCA Quantification Assay or other method of choice to determine protein concentration. 

 



SECTION: RNase and DNase Treatment of Lysates
3. RNase and DNase Treatment of Lysates
Perform these steps while beads are coupling to antibodies. 
 

 

SECTION: RNase and DNase Treatment of Lysates
3.1. Make serial dilutions to achieve desired RNaseI dilution then add 10 µl of RNaseI and 2 µl of Turbo DNase to cell lysates
SECTION: RNase and DNase Treatment of Lysates
3.2. Digest RNA for exactly 3 minutes at 37°C shaking at 1100 rpm then immediately transfer to ice and incubate for another 3 minutes. 

 

SECTION: RNase and DNase Treatment of Lysates
3.3. Spin lysates for 10 minutes at 4°C at 16,000g then transfer lysates to a fresh tube. 
 4 °C 10 min 

SECTION: Immunoprecipitation and Washes
4. Immunoprecipitation and Washes
Keep the timing of IP consistent between experiments as RNaseI remains in samples and has residual activity even at 4°C. 
SECTION: Immunoprecipitation and Washes
4.1. Set up 50 µl of RNase-treated lysate with 10 µl of antibody-bead mixture in 6X Protease Inhibitor and incubate for 2 hours at 4°C. 
4 °C 120 min 

SECTION: Immunoprecipitation and Washes
4.2. Magnetically attract beads and remove supernatant.

 

SECTION: Immunoprecipitation and Washes
4.3. Wash 2 x in 200 µl High Salt Buffer and keep the second wash for at least 1 minute at 4°C.
SECTION: Immunoprecipitation and Washes
4.4. Wash 2 x in 200 µl PNK Wash Buffer and keep in PNK Wash Buffer until ready to proceed. 
SECTION: Dephosphorylation and first adapter ligation
5. Dephosphorylation and first adapter ligation 
SECTION: Dephosphorylation and first adapter ligation
5.1. Prepare PNK reaction and add 20 µl per sample. 

 

SECTION: Dephosphorylation and first adapter ligation
5.2. Incubate at 37°C for 20 minutes with interval mixing in ThermoMixer.
  
 

SECTION: Dephosphorylation and first adapter ligation
5.4. Prepare Ligation mix and add 20 µl per sample. 

 

SECTION: Dephosphorylation and first adapter ligation
5.5. Incubate at 16°C overnight or at 25°C for 75 minutes with interval mixing in ThermoMixer. 

    

SECTION: Dephosphorylation and first adapter ligation
5.3. Remove PNK reaction and wash once in 200 µl PNK Wash Buffer. 
SECTION: Purification of RNA-protein complexes
6. Purification of RNA-protein complexes
Purification via SDS-PAGE can be omitted to enable a 2-day workflow (see "Omission of PAGE purification"). Please refer to the critical discussion of this step in Ernst et al. (2023) before choosing this workflow. 

SECTION: Purification of RNA-protein complexes
6.1. Remove ligation reaction and wash twice in 200 µl High Salt Buffer and twice in 200 µl PNK Wash Buffer. 

 


SECTION: Purification of RNA-protein complexes
6.2. Resuspend in 20 µl 1X LDS sample buffer containing 5% ß-mercapto-ethanol. 
SECTION: Purification of RNA-protein complexes
6.3. Denature at 70°C for 1 minute then proceed to PAGE purification. 
SECTION: Purification of RNA-protein complexes
6.4. Resolve RNA-protein complexes on NuPAGE 4-12% Bis-Tris Gel at 180V for 60 minutes.

SECTION: Purification of RNA-protein complexes
6.5. Transfer onto nitrocellulose in 1X NuPAGE transfer buffer with 10% methanol at 30V for 2 hours at RT. 

SECTION: Purification of RNA-protein complexes
6.6. Scan nitrocellulose membrane on Licor infrared scanner with 169 µm resolution. 

 

SECTION: Purification of RNA-protein complexes
6.7. Place nitrocellulose membrane on filter paper soaked in PBS to cut out region of interest and place nitrocellulose pieces in 1.5 ml LoBind Tubes.

 

SECTION: RNA Purification
7. RNA Purification

Upon capture on oligo(dT) beads (Step 7.5), resuspend beads throughout all steps by vortexing unless otherwise stated. Depending on the reaction, oligo(dT) beads can be sticky and mixing by pipetting can cause unnecessary loss of material through retention in pipette tips. 
SECTION: RNA Purification
7.1. Add 200 µl of Proteinase K buffer containing 100 µg of Proteinase K to LoBind tube containing nitrocellulose pieces and incubate at 50°C at 800 rpm for 45 minutes.
  

 


SECTION: RNA Purification
7.2. Meanwhile, prepare 10 µl of oligo(dT) beads per sample and wash once in 1 ml oligo(dT) Binding Buffer.
SECTION: RNA Purification
7.3. Resuspend oligo(dT) beads in 50 µl Binding Buffer per sample and distribute in fresh PCR tubes.
SECTION: RNA Purification
7.4. Transfer Proteinase K reaction to oligo(dT) beads and incubate at RT for 10 minutes rotating.
SECTION: RNA Purification
7.5. Wash twice in 125 µl oligo(dT) Wash Buffer and once in 20 µl 1X First-Strand (FS) Buffer by vortexing. 
SECTION: Reverse Transcription
8. Reverse Transcription and RNA elution
SECTION: Reverse Transcription
8.1. Prepare RT reaction and add 10 µl to beads - vortex to mix. 

 

SECTION: Reverse Transcription
8.2. Incubate at 50°C for 15 minutes with interval mixing then heat up to 96°C on thermomixer. 

 

SECTION: Reverse Transcription
8.3. When 96°C is reached, vortex for 30 seconds on heatblock, spin down, and immediately place on magnet on ice.
SECTION: Reverse Transcription
8.4. Remove supernatant and wash beads once in 60 µl oligo(dT) Wash buffer and once in 20 µl 1X T4 RNA Ligase buffer - vortex to mix. 

 

 

SECTION: Second adapter ligation
9. Second adapter ligation
SECTION: Second adapter ligation
9.1. Add 5 µl of 5' adapter mix to beads and vortex to mix. 

 

 

SECTION: Second adapter ligation
9.2. Incubate at 75°C for 2 minutes then immediately place on ice. 
SECTION: Second adapter ligation
9.3. Add 4 µl of Ligation mix at the top of the tube, spin down and vortex to mix. 

 

 

SECTION: Second adapter ligation
9.4. Add 10 µl PEG8000, spin down and vortex, then resuspend beads by pipetting up and down 10X at slow speed. 
SECTION: Second adapter ligation
9.5. Incubate at 37°C for 20 minutes, then cool down to 20°C. 
37 °C 20 min 

SECTION: Second adapter ligation
9.6. Add 1 µl of T4 RNA Ligase (High Concentration) and pipette to mix before incubating overnight at 20°C. 
  20 °C 

SECTION: cDNA pre-amplification
10. cDNA pre-amplification
SECTION: cDNA pre-amplification
10.1. Add 100 µl of oligo(dT) Wash buffer to ligation reaction and place on magnet. 
 

SECTION: cDNA pre-amplification
10.2. Discard supernatant and wash once more with 100 µl oligo(dT) supernatant and once in 20 µl 1X Phusion HF Buffer - vortex to mix. 
SECTION: cDNA pre-amplification
10.3. Prepare cDNA pre-amplification mix and resuspend beads in 25 µl - vortex to mix. 

 

SECTION: cDNA pre-amplification
10.4. Amplify with the following programme: 
1. 98°C - 30 seconds
        2. 98°C - 10 seconds
        3. 65°C - 30 seconds
        4. 72°C - 30 seconds
            Go to Step #2 6 times
5. 72°C - 3 minutes
6. 16°C - HOLD

 

SECTION: cDNA pre-amplification
10.5. Wash 1-2 µl of oligo(dT) beads per sample in oligo(dT) Binding buffer and resuspend in 5 µl oligo(dT) binding buffer per sample. 
SECTION: cDNA pre-amplification
10.6. Add 5 µl of oligo(dT) beads to PCR reaction and rotate at RT for 5 minutes. 
SECTION: cDNA pre-amplification
10.7. Place on magnet and transfer amplified cDNA in supernatant to a fresh tube.

 


SECTION: cDNA size selection
11. cDNA size selection


SECTION: cDNA size selection
11.1. Perform ProNex Size Selection using 2.8X ProNex beads - add 84 µl of ProNEX beads and mix by pipetting up and down 10 times.

 

SECTION: cDNA size selection
11.2. Incubate for 10 minutes at RT then place on magnet for 5 minutes. 
SECTION: cDNA size selection
11.3. Discard supernatant and wash twice in 200 µl Wash Buffer with beads remaining on the magnet. 
SECTION: cDNA size selection
11.4. Remove Wash buffer and air-dry the beads on magnet until pellet starts to show cracks. 

SECTION: cDNA size selection
11.5. Resuspend in 23 µl H2O and incubate for 5 minutes at RT. 
SECTION: cDNA size selection
11.6. Place on magnet and transfer 20 µl of supernatant to a new tube. 

 

SECTION: PCR amplification
12. PCR amplification
Estimate the necessary number of PCR cycles for ideal library amplification by running a test qPCR on 1 µl of pre-amplified cDNA and subtract 3-4 cycles from the obtained Ct value for the final library amplification. Overamplification of TLC-CLIP libraries should be avoided to minimise the number of PCR duplicates and increase the final yield of usable reads. 
SECTION: PCR amplification
12.1. Prepare qPCR Mastermix and add 9 µl to 1 µl of cDNA. 

 
Determine optimal cycle number by running the following programme:
1. 98°C - 30 seconds
            20 cycles of:
        2. 98°C - 10 seconds
        3. 68°C - 30 seconds
        4. 72°C - 30 seconds


 

 

SECTION: PCR amplification
12.2. Mix 10 µl of cDNA with 30 µl of PCR Mastermix and amplify with the following programme: 
 
 

1. 98°C - 30 seconds
            # of cycles:
        2. 98°C - 10 seconds
        3. 68°C - 30 seconds
        4. 72°C - 30 seconds
5. 72°C - 3 minutes
6. 16°C - HOLD


 

 

SECTION: Size-selection of libraries
13. Size-selection of libraries
SECTION: Size-selection of libraries
13.1. Perform ProNex Size Selection using 1.8X ProNex beads - add 72 µl of ProNEX beads and mix by pipetting up and down 10 times.
SECTION: Size-selection of libraries
13.2. Incubate for 10 minutes at RT then place on magnet for 5 minutes. 
SECTION: Size-selection of libraries
13.3. Discard supernatant and wash twice in 200 µl Wash Buffer while on magnet. 
SECTION: Size-selection of libraries
13.4. Air-dry the beads on magnet until pellet starts to show cracks
SECTION: Size-selection of libraries
13.5. Resuspend in 17.5 µl H2O and incubate for 5 minutes at RT. 
SECTION: Size-selection of libraries
13.6. Place on magnet and transfer 15 µl of supernatant to a new tube. 
SECTION: Quality Control and Quantification
14. Quality Control and Quantification

Prepare 1:3 or 1:5 dilution for QC and quantification.
SECTION: Quality Control and Quantification
14.1. Run 1 µl on Agilent High Sensitivity DNA Chip.

 

SECTION: Quality Control and Quantification
14.2. Quantify libraries using Qubit High Sensitivity dsDNA kit. 
SECTION: Multiplexing and sequencing
15. Multiplexing and sequencing

Multiplex samples in the final pool at the desired ratios and sequence on Illumina NextSeq500 using the High Output Kit for 75 cycles with the addition of 5% PhiX. 
SECTION: Demultiplexing and Trimming
16. Demultiplexing and Trimming of Reads using Flexbar

This is done in a two-step approach for the following reason:
The easiest way to trim 3' adapter contamination is to specifcy the entire adapter sequence (see Flexbar_2 below) with 'N' being used for the i7 index positions so that this step does not have to be run individually for different i7 indeces.

However, if this is specified together with --umi-tags, any bases on the 3' end that might correspond to the i7 index will be added to the read header and appended to the UMI which interferes with umi-tools as some UMIs will have more characters than others.

As such it is easiest to handle the demultiplexing based on In-read barcodes as well as the UMIs in the first step, and then trim remaining homopolymers at the 5' end as well as adapter contamination at the 3' end in a second step. 
SECTION: Demultiplexing and Trimming
16.1. Demultiplexing based on In-read barcodes

Flexbar.v3.4 was used. 
This command demultiplexes based on in-read barcodes and moves UMIs in the read header. 

-r sample.fastq.gz                # Fastq file demultiplexed based on i7 index but not trimmed
-b TLC_barcodes.fasta        # Fasta file specifying in-read barcodes and UMIs
--barcode-unassigned        # Generates file containing all unassigned barcodes
--barcode-trim-end LTAIL   # Defines barcode position within the read
--barcode-error-rate 0        # Determines number of mismatches and indels allowed in barcode
--umi-tags                            # Wildcard character 'N' specified within barcode fasta file will be appended to read name separated by underscode
-n 4                                       # Number of threads
-t path/sample_demult        # Prefix of output files
-z GZ                                    # Output files are compressed using gzip
 


Example of TLC_barcodes.fasta file
 


SECTION: Demultiplexing and Trimming
16.2. Trimming of adapter and homopolymers

flexbar.v3.4 was used
This command trims remaining homopolymers at the 5' end of reads as well as adapter contamination at the 3' end. 

-r sample_demult.fastq.gz    # Demultiplexed fastq files from previous trimming step
--adapter-seq 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG'
                                                # Seqeunce of the Illumina Multiplexing Index Read Sequencing Primer with 'N' designating the position of different i7 indices.
--adapter-trim-end RIGHT   # Defines which end of the read to trim the adapter sequence off
--adapter-error-rate 0.1        # Determines how many mismatches and indels are allowed for the adapter sequence to be removed
--adapter-min-overlap 1       # Minimum required overlap for adapter to be removed
--min-read-length 18            # Discards reads shorter than 18 nucleotides after trimming
-n 2                                         # Number of threads       
--htrim-left T                         # Trims poly(T) from the left side of the read
--htrim-max-length 2           # Defines maximum length of poly(T) stretch to be trimmed as 2
--htrim-min-length 1             # Defines minimum length of poly(T) stretch as 1
-t path/sample_tr1-2             # Prefix of output files
-z GZ                                      # Output files are compressed using gzip


 

Remove space from read header before mapping to keep UMI
 


SECTION: Mapping and Deduplication
17. Mapping is performed with STAR, keeping only uniquely mapping reads and removing the penalty for opening deletions or insertions.
SECTION: Mapping and Deduplication
17.1. STAR mapping
STAR v.2.7.3a was used

--runThreadN 8                                    # Number of threads
-genomeDir /genome_index/STAR/    # Path to genome directory
--readFilesIn sample_tr1-2.fastq.gz    # Path to fastq file
--readFilesCommand zcat                   # Uncompession Command for compressed fastq
--outSAMtype BAM SortedByCoordinate # Output sorted by coordinate
--limitBAMsortRAM 20000000000     # Maximum available RAM for sorting BAM
--outSAMattributes All                         # Include all attributes
--outSAMunmapped Within                 # Output unmapped reads within the main SAM file
--runRNGseed 42                                 # random number generator seed
--outTmpDir {localdir}                          # path to temporary directory
--outFilterMultimapNmax 1                  # maximum number of multiple alignments allowed for a read
--outSJfilterReads Unique                    # Only uniquely mapping reads are considered for splice junction output
--alignEndsType Extend5pOfRead1    # Fully extend only the 5' end of the read, local alignment for 3' end
--scoreDelOpen 0                                 # Remove deletion open penalty
--scoreInsOpen 0                                 # Remove insertion open penalty
--outFileNamePrefix path/sample       # Prefix for output files
 




SECTION: Mapping and Deduplication
17.2. Deduplication with umi-tools
UMI-tools v.1.0.1 was used in dedup mode:

-I mapping/star/sample_tr1-2.bam             # mapped bam file to read
-L logs/umi_tools/umi_tools_sample.log    # file with logging information
-S mapping/star_dd/sample_dd.bam        # output file 
--extract-umi-method read_id                   # Barcodes are contained at the end of the read header
--method unique                                         # Reads group share the exact same UMI
--spliced-is-unique                                     # Two reads that start in the same position on the same strand and having the same UMI are considered unique if one is spliced and the other is not.