MAPCap: A fast and quantitative transcription start site profiling protocol

Below we present a simple and quick TSS quantification protocol, MAPCap ( M ultiplexed A ffinity P urification of Cap ped RNA) that enables users to combine high-resolution detection of transcription start-sites and differential expression analysis. MAPCap can be used to profile TSS from dozens of samples in a multiplexed way, in 16-18 hours. MAPCap data can be analyzed using our easy-to-use software icetea (https://bioconductor.org/packages/icetea), which allows users to detect robust TSS using replicates, and perform differential TSS analysis.


Introduction
The MAPCap protocol described below is discussed in detail in our manuscript: MAPCap allows high-resolution detection and differential expression analysis of transcription start sites (Bhardwaj and Semplicio et al., Nature Communications. 2019).
Please find the full protocol, along with the sequence of oligos, PCR primers and ERCC controls as the attached file (pdf).

Reagents
Unless indicated otherwise, all buffers were prepared as a stock solution with RNase-free water in freshly autoclaved bottles. Buffers are decanted into Falcon tubes for immediate use and only pipetted out of them. Unless indicated otherwise, all buffers are kept at 4ºC and Falcons with aliquots of buffers are kept on ice.

Generation of capped ERCC spikes
Ten spike sequences were chosen from the ERCC RNA Spike-In Mix and PCR primers were designed to produce ~500 bp long DNA fragments (Table 1). At the 5' end, we inserted a T7 class II promoter ɸ 2.5 which creates more homogenous 5' end transcription promoter sequences. The general PCR forward primer was designed to anneal to the T7 transcription site while the individual reverse primers anneal to the respective ERCC spike sequence. 1) Reverse transcribe 1 µg of ERCC RNA Spike-In Mix with random hexamers using SSIII according to manufacturer's instruction.
2) Top-up to 50 µl volume with RNase-free water.
3) Perform PCR with ~10 ng of reverse transcribed ERCC spike mix as template.

RNA Preparation
Preparing the beads for IP 1. Wash 25 µl of Dynabeads ProteinG slurry magnetic beads per sample with IPP buffer and resuspend in at least 300 µl IPP buffer. (e.g. For 12 samples we used 300 µl of beads, which after washing were resuspended in 1 ml of IPP buffer.) 2. For each IP, we use 2.5 µg of anti-m7G antibody.
3. Add the antibody to the beads in IPP and incubate at 4ºC rotating for at least 1 hour.
4. Wash the beads three times with IPP buffer, resuspend in IPP buffer and make aliquots of 150 µl per sample

Preparing total RNA for IP
Capped RNAs within the cell mainly consist of snRNAs and snoRNAs. To avoid losing sequencing power and increase detection sensitivity, we have therefore developed a targeted depletion approach using DNA antisense oligos followed by RNase H treatment.
These oligos were designed to bind within the first 40 nt of small abundant capped RNAs.
The most abundant sn-and snoRNAs based on a first run without depletion were chosen as targets ( Table 2). The thereby resulting 20-40 nt long capped RNA species are then removed through column purification using the RNA Clean and Concentrator Kit.
rRNA species do not contain a cap and are therefore excluded from the IP. However, due to their abundance, it is possible that a substantial portion of rRNAs can stick to the 8 beads. To reduce the number of reads coming from rRNAs, we implemented a similar approach for rRNA removal as for the abundant RNAs (Table 3). In addition (also possible as a stand alone solution) we treat the RNA sample with Terminator Exonuclease, which degrades RNA species with a single phosphate group at their 5' end.

Optional: polyA RNA isolation
If the desired RNA species contains a polyA tail, we recommend a polyA RNA isolation prior to performing MAPCap. This is the most effective way to remove unwanted capped and/or abundant RNA species (sn-, sno-and rRNAs). Starting from polyA RNA, you can directly continue from the fragmentation step of the procedure.

Optional: Adding capped RNA spike-ins
Add 0.05% of capped RNA spike master mix to each sample. E.g. for an RNA starting amount of 5 µg, add 2.5 ng of capped RNA spike master mix.
2. Add 12 µl of AS-master mix to each sample, incubate the RNA at 70ºC for 2 min followed by gradual cooling to 37ºC (for this, we changed the temperature of the thermocycler from 70ºC to 37ºC. This takes around 10 min.) 3. Once the samples have reached 37ºC, add 1 µl of RNase H (2 U/µl) and incubate at 37ºC for 30 min at 1100 rpm interval shaking (30 s on, 120 s off). 9 4. Incubate the samples at 70 ºC for 5 min, put on ice immediately for at least 1 min, and add 2 µl of 5' Terminator Exonuclease (1 U/µl

CRITICAL: It is important to obtain RNA of a uniform size of around 200-400 nt in order to
achieve ideal conditions for sequencing. We recommend testing the fragmentation in advance with several conditions (Figure 1).

IP
1. Transfer the sonicated RNA in 100 µl TE buffer to the 150 µl aliquots of antibody coupled beads.

Resuspend the mix and incubate at 4ºC for 1-2 hours.
General remarks regarding washing beads: • To properly remove all liquid from the beads for washing and resuspension in a new solution, we perform a short spin to remove any liquid that has accumulated in the lid of the tube.
• We then place the tube on a magnet and wait until the beads are properly bound before removing the solution with a vacuum pump or manual pipetting.
• After the last removal of the washing buffer, we take the tubes from the magnet and perform another quick spin on a table centrifuge before placing the tubes back on the magnet. This allows us to collect the remaining liquid at the bottom of the tube with a 10 µl pipet.

General remarks regarding resuspension of beads in a new reaction mix:
• Most reactions are carried out in 20 µl volume. It is therefore important to remove all excess volume from the wash step before continuing with the protocol (see general remarks on washing, point 3).
• We elute beads by dispensing the 20 µl from the wall of the tube where the beads were fixed to the side. Repeat this step until all beads are off the wall and gently resuspend until the beads are homogeneously in solution.

Library preparation
1. Wash the beads 3 times with IPP buffer. Add at least 500 µl of IPP buffer and rotate the beads at 4ºC for 5 min.

Library amplification
In order to perform as few PCR amplification cycles as possible, we recommend performing a qPCR on the circularized DNA template and compare the cycle numbers to a standard library. The standard library is a previously sequenced MAPCap library where the concentration is known. We aim at a library of around 2-4 ng/µl in 10 µl final volume after PCR and Ampure beads clean-up. We use 1 µl of circularized cDNA which is serially diluted 14 3 times to get different measurements with different concentrations. Typically, MAPCap requires between 14 and 18 cycles of PCR depending on the starting amount used. 3. Clean-up the amplified library with two rounds of Ampure beads at a 1x ratio.
○ Equilibrate Ampure XP beads at room temperature for 30 min and vortex the beads well before use. ○ Add 40 µl (1x ratio) of Ampure beads to the library and mix well. ○ Incubate 5 min at room temperature before placing the tube on a magnet. ○ Once beads are fixed to the magnet and the solution is clear, remove 75 µl of the liquid.
(Caution: Do not disturb the beads.) ○ Wash 2x with 200 µl 80% EtOH without removing the beads from the magnet. ○ Dry the beads at room temperature while on the magnetic stand for 5 min (do not overdry).
○ Remove the tube from the magnet and resuspend the beads in 20 µl of RNase-free water.
○ Reclaim beads on a magnet and collect the sample in a new tube. ○ Add 20 µl (1x ratio) of Ampure beads to the library and mix well.