TITLE: Low Quantity single strand CAGE protocol
AUTHORS: Hazuki Takahashi, Hiromi Nishiyori-Sueki, Piero Carninci

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The </span><span style = ":UNDERLINE;">C</span><span>ap </span><span style = ":UNDERLINE;">A</span><span>nalysis of </span><span style = ":UNDERLINE;">G</span><span>ene </span><span style = ":UNDERLINE;">E</span><span>xpression (CAGE) is a powerful method to identify the Transcription Starting Sites (TSSs) of capped RNAs while simultaneously measuring transcript level. CAGE allows mapping at single nucleotide resolution all active promoters and enhancers. Large CAGE datasets have been over the years by individual labs and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation Of the Mammalian Genome (FANTOM). These dataset constitute open resource for TSS annotations and gene expression analysis. </span></div><div class = "text-block">Here we provide a protocol for the most recent CAGE version of the method, which is called Low Quantity (LQ) single strand (ss) CAGE (LQ-)ssCAGE. The methods allow to profile samples starting from low quantity of RNAs. Samples may typically include cells cultured in small volumes to maximizing large scale perturbations, cellular compartments like nuclear RNAs or samples from developmental stages. The advantage of (LQ-)ssCAGE is to allow working with small samples and simultaneously avoiding PCR amplification: this step help to preserve exquisite quantification of expression. The protocol makes use of simplified steps, by loading single stranded cap selected cDNA. Further, is allows reducing the RNA amount down to 25 ng and to shorten preparation time (less than 3 days) and to prepare libraries parallel, increasing the number of samples that one operator can reasonably handle (up to 96 samples). By proper organization of the flow of libraries preparation, an operator could further scale up the throughput. The obtained libraries are suitable for sequencing by various Illumina sequencing platforms using indexed reads and original barcode identifiers.</div></div>

[STEPS]
?. 
?. Reverse Transcription (RT) to generate RNA/cDNA hybrids
?. Oxidation to modify diol group of cap structurea. Mix 40 µL of RNA/cDNA hybrid from step 1.1e. or 1.2g., 2 µL of 1 M NaOAc (pH 4.5) and 2 µL of 250 mM NaIO4 in a tube by 10 times pipetting.b. Incubate for 5 min on ice in dark by aluminum foil wrapping.c. Add 16 µL of 1 M Tris-HCl (pH 8.5) to neutralize the solution and mix well by pipetting.
NOTE: 1.5 mL tube, 8-Strip PCR tube or16 wells plate can be used depending on how many samples should be prepared.
?. 
?. single strand CAGE (ssCAGE)When the amount of RNAs is more than1 µg, choose this RT protocol. Here is an example of 5 µg starting RNAs.When the amount of RNAs is less than 1 µg, go to step 1.2.a. Mix 5 µL of RNAs (1 µg/µL), 1 µL of RT primer (N6+TCT) and 4 µL of RNase free water in a tube.b. Incubate the RNA/primer mix from step a. at 65 °C for 5 min and immediately put on ice for 2 min.c. Add the following components to the solution from step b. and carefully mix by pipetting on ice. 5 x First-Strand buffer                             4 µL10 mM dNTPs                                          1 µL0.1 M DTT                                                 1 µLRNase free water                                     2 µLSuperScript III Reverse Transcriptase  2 µLd. Incubate the mixed solution (total 20 µL) at 25 °C for 30 sec, 55 °C for 30 min and keep 4 °C to generate RNA/cDNA hybrids.e. Add 36 µL (1.8-folds) of RNACleanXP beads to 20 µL of RNA/cDNA hybrids from step d. and mix well by pipetting. ①. Incubate at room temperature for 5 min. Spin down and set the tube on magnetic bar for 5 min. ②. Discard the supernatant by pipette aspiration. ③. Wash the beads with 200 µL of 70% EtOH. ④. Set the tube on magnetic bar for 5 min. ⑤. Discard the supernatant by pipette aspiration. ⑥. Repeat step ③ ~ ⑤.⑦. Discard the 70% EtOH completely by pipette aspiration. ⑧. Add 42 µL of RNase free water and mix by pipetting extensively (more than 60 times) to elute RNA/cDNA hybrids.⑨. Incubate at room temperature for 5 min. ⑩. Spin down and set the tube on magnetic bar for 5min. ⑪. Transfer supernatant (~ 40 µL of the cDNA/RNA hybrids) to new tube.
NOTE: Use 1.5 mL tube, 8 strip PCR tube or 16-96 wells plate  depending on numbers of samples.
?. Low Quantity single strand CAGE (LQ-ssCAGE)When the amount of starting RNAs is less than 1 µg, choose this protocol. a. Mix 4 µL of 50 ng RNAs (12.5 ng/µL) and 1 µL of 1.25 mM RT primer containing barcode in a 96 wells plate by pipetting. b. Incubate the RNA/primer mix form step a. at 65 °C for 5 min and immediately put on ice for 2 min.c. Mix the following components (enzyme mix) on ice: 5 x First Strand buffer                             2.0 µL 0.1 M DTT                                                 0.5 µL10 mM dNTPs                                          0.5 µLRNase free water                                     1.0 µLSuperScript III Reverse Transcriptase  1.0 µLTotal volume                                             5.0 µLd. Add 5 µL of enzyme mix from premix solution at step c. to RNA/primer mix from step b. and carefully mix 10 times by pipetting on ice.e. Incubate at 25 °C for 30 sec, 50 °C for 30 min and keep 4 °C to generate RNA/cDNA hybrids.f. Mix samples by following steps. ①. Transfer each 10 µL of RNA/cDNA hybrids from step e. to one of new 1.5 mL tube (total volume is 480 µL). ②. Add 15 µL of RNase free water to the first 8 wells at the 96 wells plate from step ①, wash wells by pipetting, and transfer the 15 µL of the solutions to the next 8 wells. ③. Wash the 8 wells by pipetting and transfer 15 µL of the solutions to the next 8 wells. ④. Repeat step ②three times until the end of 8 wells and transfer all solutions (total volume is 120 µL (15 µL x 8 wells)) to the 1.5 mL tube from step ①(final volume is 600 µL).⑤. Mix 600 µL of the solution from step ④by vortex, spin down and aliquot 200 µL in new two tubes of 1.5 mL tube (total 200 µL in 3 tubes of 1.5 mL tube). g. Add 300 µL (1.8 -folds) of RNAClean XP beads to the 48 mixed RNA/cDNA hybrids in a 1.5 mL tube from step f.⑤, mix well by pipetting and then elute the mixed RNA/cDNA hybrids by following steps.①. Incubate at room temperature for 10 min, spin down and set the 1.5 mL tube on magnetic bar for 5 min. ②. Discard the supernatant by pipette aspiration. ③. Wash the beads with 1.2 mL of 70% EtOH. ④. Set the 1.5 mL tube on magnetic bar for 5 min. ⑤. Discard the supernatant by pipette aspiration. ⑥. Repeat step ③~ ⑤ twice.⑦. Discard the 70% EtOH completely by pipette aspiration. ⑧. Add 100 µL RNase free water and mix by pipetting extensively (more than 60 times) to elute RNA/cDNA hybrids.⑨. Incubate at room temperature for 5 min. ⑩. Spin down and set the tube on magnetic bar for 5min. ⑪. Transfer 100 µL of the cDNA/RNA hybrids to new 1.5 mL tube. ⑫. Repeat step ⑧ ~ ⑪ twice (final volume is 200 µL). ⑬. Concentrate 200 µL of the cDNA/RNA hybrids solutions from step ⑫ to around 40 µL by SpeedVac vacuum concentrator at 37 °C, and collect the solutions from 3 of 1.5 mL tubes to 1 of 1.5 mL tube (total volume is around 120 µL) and concentrate to 40 µL in the 1.5 mL tube at 37°C. The timing is around 2 h.
NOTE: The amount of RNAs in one tube should be 25 ng ~ 100 ng for the RT reaction. After RT, samples have each barcode and can be mixed up to 5 µg. For instance, if the starting RNA amount is 50 ng, 100 samples can be mixed. The number of mixed samples is depending on how much samples you have and how deep the samples should be sequenced. Here is an example of 48 mix samples from each 50 ng starting RNA.CRITICAL: The mixed solution should be less than 5 µg. Otherwise the following linkers at step 9 and 11 are not enough for the linker ligation.
NOTE: Make premix solution for 48 samples x 1.1 samples.
NOTE: Step f. is for the collection of remaining molecules to avoid losing any RNA/cDNA hybrids in the wells. We recommend to aliquot less than 200 µL per each 1.5 mL tube at step f.⑤due to the volume limitations of next RNAClean XP purification step.
CRITICAL: DO NOT DRY UP the cDNA/RNA hybrids solutions by the concentrating step ⑬. Check the sample volume during the concentration several times and adjust the final volume to 40 µL with RNase/DNase-free when the volume become less than 40 µL.
?. PurificationAdd 108 µL (1.8 -folds) of RNACleanXP beads to 60 µL of oxidated RNA/cDNA hybrid from step 2, mix well by pipetting and then elute the 48 samples mixed RNA/cDNA hybrids by following steps.①. Incubate at room temperature for 5min.②. Spin down and set the tube on magnetic bar for 5 min. ③. Discard the supernatant by pipette aspiration. ④. Wash the beads with 200 µL of 70% EtOH ⑤. Discard the 70% EtOH. ⑥. Repeat step ④~ ⑤twice and discarded 70% EtOH completely.⑦. Add 42 µL of RNase free water and mix by pipetting extensively (more than 60 times) to elute supernatant. ⑧. Incubate at room temperature for 5min. ⑨. Spin down and set the tube on magnetic bar for 5 min. ⑩. Collect 40 µL of the supernatant to new tube.
?. Biotinylation by the coupling reaction to the oxidized RNA/cDNA hybridsa. Mix 40 µL of purified oxidized RNA/cDNA hybrid from step 3, 4 µL of 1M NaOAc (pH 6.3) and 4 µL of 100 mM Biotin (long arm) hydrazide by 10 times pipetting.b. Incubate for 30 min at 40 °C.c. Add 86.4 µL of RNACleanXP (1.8 -folds) to 44 µL of solution from step b, and perform purification by following step 3 ① ~ ⑩.
NOTE: Biotinylation happens both 5’end of capped RNAs and 3’end of RNA (see Figure 2 at published protocol).
?. RNaseONE treatment to digest RNA of RNA/cDNA hybridsa. Add 4.5 µL of 10 x RNaseONE buffer and 0.5 µL of RNaseONE to 40 µL of purified biotinylated RNA/cDNA hybrids from step 4 and mix 10 times by pipetting. b. Incubate for 30 min at 37 °C.c. Add 81 µL of RNACleanXP (1.8 -folds) to the solution from step b. and purify by the following step 3 ①~ ⑩.
CRITICAL: This step is critical to digest uncompleted cDNA synthesis to the 5’ end of capped RNAs (See Figure 2 at published protocol).
?. Dynabeads M-270 Streptavidin beads preparationa. Add 30 µL of Dynabeads M-270 Streptavidin to new 1.5ml tube, set on the magnetic bar for 5 min, and discard supernatant. b. Wash the beads with 30 µL of LiCl buffer and discard supernatant.c. Repeat step b. for 2 times.d. Resuspend the beads in 35 µL of LiCl buffer.
?. CapTrap reactiona. Add 35 µL of beads from step 6 to 40 µL of RNA/cDNA hybrids from step 5 and mix well by pipetting.b. Incubate for 15 minutes at 37 °C and mix by pipetting at the time of 7 min. c. Spin down and set the plate on the magnetic bar for 2 min and discard the supernatant by the pipette aspiration.d. Add 150 µL of TE wash buffer and mix 60 times by pipetting. Spin down and set the plate on the magnetic bar for 2 min and discard the supernatant. e. Repeat step d. for 3 times.f. Add 35 µL of Release buffer to the beads and mix 60 times by pipetting. g. Incubate for 5 min at 95°C for 5min and on ice for 1 min. h. Spin down and set the tube on the magnetic bar for 2 min.i. Transfer the supernatant containing CapTrapped cDNA from step h. to new 16 well plate. j. Add 30 µL of Release buffer to the beads and mix well by pipetting, spin down, and set the tube on the magnetic stand for 2 min. k. Transfer 30 µL of supernatant containing CapTrapped cDNA to new tube.
?. RNaseONE and RNaseH reaction to remaining RNAsa. Add 2.9 µL of release buffer, 2 µL of RNaseONE and 0.1 µL of RNase H to 30 µL of CapTrapped cDNA from step 7 and mix 10 times by pipetting.b. Incubate at 37°C for 30 min.c. Add 126 µL of AMPureXP (1.8 -folds) to 33 µL of cDNA from step b. and perform purification by following step 3 ① ~ ⑩.d. Dry up 40 µL of purified CapTrapped cDNA by the SpeedVac concentrator at 37°C for around 75 min. e. Add 4 µL of RNase free water to the dried pellet.
?. 5' Single Strand Linker Ligationa. Incubate 4 µL of cDNA at 95 °C for 5 min and put on ice for 2 min. b. Incubate 4 µL of 5’ linker at 55 °C for 5 min and put on ice for 2 min. c. Mix 4 µL of 5’ linker, 4 µL of CapTrapped cDNA from step a. and 16 µL of Mighty Mix by pipetting (total volume is 24 µL). d. Incubate at 16 °C for 16 h (overnight).
?. Purification to remove excess 5’ linkers and linker dimersa. Add 46 µL of RNase free water and 126 µL of AMPureXP (1.8 -folds) to 24 µL of cDNA from step 9, mix well by pipetting.b. Purify the solution by following step 3 ①~ ⑩.c. Incubate at 95 °C for 5 min and immediately put on ice for 2 min.d. Add 48 µLof AMPureXP beads (1.2 -folds) to 40 µL of cDNA and mix well by pipetting. e. Repeat purification by following step 3 ① ~ ⑩.f. Dry up 40 µL of cDNA by SpeedVac concentrator at 80°C for around 35 min. g. Add 4 µL of RNase free water to the dried pellet.
CRITICAL: It is important to perform heat inactivation and repeat purification step to remove non-ligated excess linkers during the purification step. Otherwise, these excess linkers can disturb library sequencing quality; excess linkers compete with the library to anneal Illumina flow-cells. In addition, 5’linker-3’linker dimers can be created at the next step 11.
?. 3’ Single Strand Linker Ligationa. Incubate 4 µL of cDNA at 95 °C for 5 min and put on ice for 2 min. b. Incubate 4 µL of 3’ linker at 65 °C for 5 min and put on ice for 2 min. c. Mix 4 µL of 3’linker, 4 µL of cDNA from step a. and 16 µL of Mighty Mix by pipetting. d. Incubate at 30 °C for 4 h.
NOTE: When you have more than 2 libraries with same barcode at RT primer and plan to mix libraries at the sequencing step 14, use different Index at the 3'linker.
?. Purification to remove excess 3’ linkers and linker dimers (final library)a. Purify the solution from step 11 by following step 11a.-f.b. Dissolve the pellet with 10 µL of RNase, DNase-free water.
?. Library QCQuantify the concentration of library by KAPA Library Quantification Kit.
?. Sequencinga. Mix the following components Library (2250 amol)                    X µLRNase, DNase-free water  (19-X) µL2N NaOH                                       1 µLTotal                                             20 µLb. Incubate at room temperature for 5 min.c. Put the tube on ice and add 20 µL of 1 M Tris-HCl (pH 7.0, pre-chilled) to neutralize.d. Add 110 µL of HT1 buffer.e. Transfer 120 µL of the denatured and diluted library to HiSeq2500.Sequencing kit  ssCAGE: 50 bp Single Read sequencing.LQ-ssCAGE: 50 bp Paired-End sequencing (see Figure 1 for the library structure).       Read 1: to read sequence information of cDNA        Read 2: to read sequence information of barcode in RT primer       Index 1: to read sequence information of index at the 3'linker