TITLE: FLASH
AUTHORS: Ibrahim Ilik, Tugce Aktas, Daniel Maticzka, Rolf Backofen, Asifa Akhtar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Determination of the </span><span style = "font-style:italic;">in vivo</span><span> binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding site has not been described to date. Here we report FLASH (</span><span style = ":UNDERLINE;">F</span><span>ast </span><span style = ":UNDERLINE;">L</span><span>igation of RNA </span><span style = ":UNDERLINE;">a</span><span>fter </span><span style = ":UNDERLINE;">s</span><span>ome sort of Affinity Purification for </span><span style = ":UNDERLINE;">H</span><span>igh-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein-RNA interactions in living cells. The entire FLASH protocol, starting from cells-on-plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions </span><span style = "font-style:italic;">in vivo</span><span>. </span></div></div>

[STEPS]
?. [Lysate preparation]
Crosslink cells with with UV-C (0.15 - 0.2 mJ/cm2) irradiation.Type, amount, state of the cells varies depending on the intended application. As a rule of thumb, crosslink cells in 15cm-plates on water/ice and as close to the lamp as possible (3-6 cm distance). Wash the cells with ~6mL of ice-cold PBS, and add 6mL of ice-cold PBS again before crosslinking. It is important to do at least one wash of PBS to remove UV-absorbing media components (phenol red, nucleotides etc.). We usually use half of one 15cm-plate for one IP/pull-down. In a typical experiment, two 15cm plates are crosslinked, and half of the cells from each plate is used as a biological replicate pair, and the other two are kept as backup. The cells, if they are not used immediately, are spun down at 1000g for 1 minute, and snap-frozen with liquid nitrogen after removal of PBS, and kept at -80˚C until use.
?. [Lysate preparation]
Re-suspend the cell pellet with , sonicate with Bioruptor Plus (5 cycles, LOW,  ON,  OFF).  Spin-down the lysate @ 20.000g for , use the supernatant for IP/pulldown.There are a few important points here. NLB (short for Native Lysis Buffer) is used here for cell disruption, because it is a relatively high-salt buffer (~500mM NaCl) with high amounts of non-denaturing detergents (1% Triton-X, 0.1% Tween-20). Combined with the Bioruptor treatment, this homogenization/disruption method is very effective in solubilizing target proteins. It is advisable to check if the target is indeed solubilized properly before proceeding with the rest of the protocol.Other types of sonication/homogonization may be possible, and must be tested empirically. We do not recommend using Bioruptor Pico for this purpose.
[1xNLB]
?. [Polyhistidine Purification]
Add the lysate to 25µL of His-Tag Isolation and Pulldown beads. Take 25µL of nicely resuspended beads (resuspending either by vortexing, or preferably by rotating the beads end-to-end in the cold-room until no clumps are visible on the walls), wash them once with 1xNLB, resuspend with  NLB. Add the lysate to this suspension, pipette up and down 2-3 times then incubate for 5-10 minutes on ice. Rotation is not necessary, these beads do not settle in this time-frame. Wash the beads 1x with NLB.Many researchers are very sceptical about poly-histidine purifications, because they tend to be “dirty”. This is indeed true, single-step poly-his purifications are generally quite dirty. What we do here however is a bit different: these beads almost quantitatively collect the target protein only after 5 minutes of incubation. This is quite extraordinary, and coupled to a second purification (via the Biotin group in this protocol) leads to very clean purifications. The speed of the first pull-down probably helps to remove most of the extremely abundant RNAs that tend to be absorbed to bead surfaces during extended purifications.
500 µl
?. [Polyhistidine Purification]
Elute with 0.5mL NLB + 250mM imidazole, 5 minutes on ice.One can use as little as 50mM imidazole, these beads release target very quickly with >50mM imidazole. The elution buffer is prepared using 2xNLB stock, 2M imidazole pH 8.0 and water.
?. [Streptavidin purification]
Incubate the eluate with 25µL MyOne™ Streptavidin C1 beads (in 500µL NLB) forin the cold-room. Wash once with HSB and once with NDB.The incubation can be as short as 30 minutes and as long as 2 hrs, this can be empirically determined for each protein. It would be best to limit the incubation to the shortest possible time to reduce non-specific RNA absorption on the beads. Other streptavidin-coupled beads may also be used, however agarose/sepharose beads should best be avoided.
?. [Partial RNase digestion]
Re-suspend the beads with 90µL NDB. Add 10µL of RNaseI diluted in NDB (1:500-1:2000). Incubate at  for . Keep on ice for a minute. Wash with HSB, then NDB.This step is very important. The more RNAse once uses, the sharper the peaks become, but the recovery is also reduced. We have used dilutions ranging from 1:400 to 1:8000. We would recommend to start with 1:2000. Due to batch effects, and other uncontrollable laboratory conditions, it is highly recommended to initially prepare a library where distinct s-oligos are used with different RNAse treated samples, which are then pooled after ligation and sequenced.
?. [Stringent washes]
Re-suspend the beads with 7µL of room-temperature, RNase-free water. Add 1µL of selected s-oligo (10µM). Add the rest of the ligation mix. Incubate @ 25˚C for 60 minutesPEG8000 and ATP is shipped with T4 RNA Ligase, when ordered from NEB. PEG8000 is extremely viscous, and that is the reason why the reaction is set up in this way (first water, then adapter, then the rest).Adapters are internally barcoded (TGTAGC below). It is possible to mix negative controls (GFP-expressing cell line, parental cell line, point mutant that is supposed to not bind RNA etc) with the sample after ligation. This allows us to evaluate background very precisely and filter false-positives efficiently.The adapters are key to the success of this protocol. They basically look like this:This adapter is ligated to RNA via T4 RNA ligase. The self-annealed part then is used as a reverse-transcription primer. At the end of reverse transcription, RNaseH is used to remove RNA and create a 5’-phosphate at the edge of the adapter. This cDNA molecule is then circularized by CircLigase (requires 5’-phosphate) and then PCR-amplified (adapter is based on the Y-shape adapter of Illumina and contains P5 and P3 sequence for amplification). We have a patent application for it too: WO/2017/013005  AB110X T4 Ligase Buffer2µL2PEG80004µL3s-oligo (10µM)1µL4Superas-IN0.5µL5T4 RNA Ligase 1 (10U/µL)1µL6ATP (1mM)2µL7H2O7 +2.5µL
AB110X T4 Ligase Buffer2µL2PEG80004µL3s-oligo (10µM)1µL4Superas-IN0.5µL5T4 RNA Ligase 1 (10U/µL)1µL6ATP (1mM)2µL7H2O7 +2.5µL
?. [Dephosphorylation of RNA-ends with T4 PNK]
Resuspend with 20µL of the MES-PNK mix. Incubate @ 37˚C for 20 minutes. Wash once with HSB, once with NDB.At this step, T4 PNK is used to dephosphorylate the 3’-ends of RNAseI-digested RNA. RNaseI leaves a cyclic 2’,3’-bisphosphate behind, which is refractory to adapter ligation. T4 PNK removes this and other phosphate groups and leaves a 3’-OH, which is suitable for ligation. NEB also sells a phosphatase-minus PNK, obviously do not use that. Any other RNase-inhibitor would work here, and addition of β-mercaptoethanol is absolutely necessary.  AB12X PNK-MES buffer (pH 6.0)10µL2SUPERase• In™ (20U/µL)0.5µL3β-mercaptoethanol (0.1M)1µL4T4 PNK (10U/µL)1µL5H2O7.5µL
AB12X PNK-MES buffer (pH 6.0)10µL2SUPERase• In™ (20U/µL)0.5µL3β-mercaptoethanol (0.1M)1µL4T4 PNK (10U/µL)1µL5H2O7.5µL
?. [s-oligo ligation]
Wash once with HSB, merge all relevant samples. Wash once with LDS-buffer, once with PLB buffer, 1x HSB, 1x NDB.Carry out LDS and PLB washes at room-temperature, and the rest in the cold-room or on ice.
?. [Removal of the 3'-phosphate from s-oligo]
Resuspend the beads with 35µL of NDB, add 1µL RNAsin+, 4µL rSAP. Incubate @ 37˚C for 15 minutes. Wash once with HSB, once with NDB.This step is necessary to remove a blocking 3’-phosphate from our s-oligo. It is there to prevent self-circularization during ligation, but has to be removed before reverse-transcription. One can also use T4 PNK, exactly as done in step (7).
?. [Deproteinization and recovery of RNA]
Resuspend the beads with the proteinase-K mix and incubate for 20 minutes at 42˚C.  AB12X ProK buffer50µL220% SDS1µL3Proteinase K (20mg/mL)10µL4H2O39µL
AB12X ProK buffer50µL220% SDS1µL3Proteinase K (20mg/mL)10µL4H2O39µL
?. [Deproteinization and recovery of RNA]
Magnetize the beads, remove the supernatant and transfer to a new tube. Use the Oligo Clean and Concentrator Kit to purify RNA (use 200µL Binding Buffer and 400µL of Ethanol for binding). Elute with 10.5µL of RNase-free water.
?. [Reverse transcription and phosphorylation of 5'-ends]
Incubate the RNA elution at fro 2 minutes, transfer immediatately on ice, leave for a minute. Add the rest of the reverse-transcription mix, prepared as per SuperScript III manual. Incubate at 42˚C, then at 50˚C, then at 55˚C, 10 minutes each.  AB110X RT Buffer2µL210mM dNTPs1µL325mM MgCl24µL40.1M DTT2µL5RNaseOUT (40U/µL)0.5µL6SSIII (200U/µL)0.5µL7RNA eluate in H2O10µL
AB110X RT Buffer2µL210mM dNTPs1µL325mM MgCl24µL40.1M DTT2µL5RNaseOUT (40U/µL)0.5µL6SSIII (200U/µL)0.5µL7RNA eluate in H2O10µL
?. [Reverse transcription and phosphorylation of 5'-ends]
In order to remove RNA and phosphorylate the 5'-ends, cool the RT reactions to room-temperature, and add 2µL of E. coli RNaseH (2U/µL), which is shipped with the SuperScript III First-Strand Synthesis System. Incubate for  at.Other sources of RNaseH can be used as long as it is an RNaseH1-type, processive RNaseH and not the RNaseH2-type which is not processive. Alternatively, RNA can be removed with NaOH, followed by T4 PNK-mediated phosphorylation of the cDNA ends. See the manuscript for details.
?. [Circularization of cDNA]
Set up the circularization reaction. Incubate at for at least .   AB110X Buffer1µL250mM MnCl20.5µL35M Betaine	2µL4CircLigaseII0.5µL5cDNA6µL6 10µL total. CircLigase can also be used instead of CircLigaseII, remember to add ATP to the reaction!
AB110X Buffer1µL250mM MnCl20.5µL35M Betaine	2µL4CircLigaseII0.5µL5cDNA6µL6 10µL total.
?. [Reverse transcription and phosphorylation of 5'-ends]
Optional: To remove any RNA that may be left in the reaction, add 2.5µL of 1N NaOH. Incubate @ 80˚C for 5 minutes. Neutralize with 25µL 0.2M Tris.
?. [qPCR to determine optimal cycling conditions]
In order to determine the number of cycles we should use to amplify the circularized cDNA, we first set up a qPCR reaction using 1µL of the CircLigase reaction. This step is highly empirical and is not meant to be used as a way to quantitate different libraries. The purpose here is to estimate a cycle number with which we are reasonably sure that we are not over- or underamplifying our libraries.  AB1FastStart Universal SYBR Green Master Mix (2X)10µL2circularized cDNA1µL3primerL (10µM):2µL4primerR (10µM):2µL5Water5µLPrimerL (410a in the manuscript): TACACGACGCTCTTCCGATCTPrimerR (410b in the manuscript): GACGTGTGCTCTTCCGATCT
AB1FastStart Universal SYBR Green Master Mix (2X)10µL2circularized cDNA1µL3primerL (10µM):2µL4primerR (10µM):2µL5Water5µL
?. [Reverse transcription and phosphorylation of 5'-ends]
In order to remove RNA and phosphorylate the 5'-ends, cool the RT reactions to room-temperature, and add 2µL of E. coli RNaseH (2U/µL), which is shipped with the SuperScript III First-Strand Synthesis System. Incubate for  at.Other sources of RNaseH can be used as long as it is an RNaseH1-type, processive RNaseH and not the RNaseH2-type which is not processive. Alternatively, RNA can be removed with NaOH, followed by T4 PNK-mediated phosphorylation of the cDNA ends. See the manuscript for details.
?. [Reverse transcription and phosphorylation of 5'-ends]
Optional: To remove any RNA that may be left in the reaction, add 2.5µL1M  NaOH. Incubate @ 80˚C for 5 minutes. Neutralize with 25µL 0.2M Tris.
?. [Reverse transcription and phosphorylation of 5'-ends]
Use the Oligo Clean and Concentrator Kit to purify cDNA (If step 15 was taken, use 100µL Binding Buffer and 200µL of Ethanol for binding, otherwise, add 28µL of water to the RT reactions first, then add 100µL of Binding Buffer). Elute with 6.5µL of RNase-free water.
?. [cDNA circularization]
Set up the cDNA circularization reaction. Incubate for at least at either in an air-incubator or in a thermal cycler with a heated-lid to prevent evaporation. We routinely leave this reaction overnight (~16hrs).CircLigase can also be used (do not forget to add ATP to the reaction in that case!).  AB110X Buffer1µL250mM MnCl20.5µL35M Betaine	2µL4CircLigaseII0.5µL5cDNA6µL6 10µL total.
AB110X Buffer1µL250mM MnCl20.5µL35M Betaine	2µL4CircLigaseII0.5µL5cDNA6µL6 10µL total.
?. [qPCR to determine optimal cycling conditions]
In order to determine the number of cycles we should use to amplify the circularized cDNA, we first set up a qPCR reaction using 1µL of the CircLigase reaction. This step is highly empirical and is not meant to be used as a way to quantitate different libraries. The purpose here is to estimate a cycle number with which we are reasonably sure that we are not over- or underamplifying our libraries.
?. [Library amplification and clean-up with AMPure XP beads]
Based on the qPCR results, determine a cycle number that will be used for PCR. In our laboratory setting, Ct-1 generally gives good results. We then use the rest of the CircLigaseII reaction to amplify our library using NEBNext® Ultra™ II Q5® Master Mix as below. The choice of amplification primers depends on the final sequencing platform that will be used, type of run.
?. [Library amplification and clean-up with AMPure XP beads]
?. [Library amplification and clean-up with AMPure XP beads]
Based on the qPCR results, determine a cycle number that will be used for PCR. In our laboratory setting, Ct-1 generally gives good results. We then use the rest of the CircLigaseII reaction to amplify our library using NEBNext® Ultra™ II Q5® Master Mix using the reaction mixture below. The choice of amplification primers depends on the final sequencing platform and type of run that will be carried out. Compatible primer sets include, but not restricted to: NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (E7600), NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1). Compatibility can be verified by cross-checking the s-oligo sequencing to the primers that will be used.   AB1NEBNext® Ultra™ II Q5® Master Mix (2X)10µL2circularized cDNA9µL3P5/i5 (10µM):1µL4P3/i7 (10µM):1µL5Water19µL
AB1NEBNext® Ultra™ II Q5® Master Mix (2X)10µL2circularized cDNA9µL3P5/i5 (10µM):1µL4P3/i7 (10µM):1µL5Water19µL
?. [Reverse transcription and phosphorylation of 5'-ends]
Use the Oligo Clean and Concentrator Kit to purify cDNA (If step 15 was taken, use 100µL Binding Buffer and 200µL of Ethanol for binding, otherwise, add 28µL of water to the RT reactions first, then continue with 100µL of Binding Buffer). Elute with 6.5µL of water.
?. [Library amplification and clean-up with AMPure XP beads]
Clean up the reaction twice with 1.5X AMPure XP beads.Add 60µL of beads to the reaction, incubate for 5 minutes. Magnetize the beads and remove the supernatant carefully. Wash twice with 500µL of freshly-prepared 70% Ethanol without resuspending the beads or removing the tubes from the magnet. Air-dry for 1-2 minutes and elute the bound DNA by resuspending the beads with 40µL of 10mM Tris pH 8.0, 0.05% Tween-20. Incubate the beads at room temperature for 1-2 minutes. Magentize the beads, transfer the eluate to a fresh tube, and repeat the purification as described above. Elute the final library with 10-20µL of 10mM Tris pH 8.0, 0.05% Tween-20.
?. [Library quantification]
Quantify the end product using Qubit or another sensitive dsDNA quantification platform. It is also generally necessary to determine the size-distribution of the library, which can be carried out with an Agilent Bioanalyzer 2100, or Fragment Analyzer or an equivalent system. Ready for sequencing!After sequencing, the UMI and the index can be found in the first 13 nucleotides of the R2 reads with this pattern: NNNNNXXXXXXNN, where XXXXXX is the index and the Ns together form the UMI.See https://clipseq.usegalaxy.eu/ for a quick introduction to CLIP-data analysis.