Sequencing library preparation

sRNA-seq library preparation was performed using NEBNext Small RNA Library Prep Set for Illumina kit (New England Biolabs [NEB]) with the following modifications. The samples were rinsed in Ca₂⁺/Mg₂⁺-free PBS and placed in 3 μL of lysis buffer containing 5 mM Tris-HCl at pH 7.5 (Sigma-Aldrich); 0.1% Tween-20 (Sigma-Aldrich); 50 mM KCl (Sigma-Aldrich), and 2.5 units of RiboLock RNase inhibitor (Thermo Fisher Scientific). One microliter of 1:3 diluted 3′ SR Adapter (NEB), 1 μL of 0.5 M KCl, 1 μL of 20 μM 5S and 5.8S rRNA masking oligo mixture, and 1 μL of nuclease-free water was added to samples. The samples were incubated in a thermal cycler for 1 min at 90°C and at for 2 min 60°C to mask the rRNA. Three microliters of 3′ ligation enzyme mix (NEB) and 10 μL of 3′ adapter ligation reaction buffer (NEB) was added to samples. The samples were incubated for 60 min at 25°C. To the samples we added 4.5 μL of nuclease-free water and 1 μL of 1:3 diluted SR RT Primer (NEB). The libraries were incubated for 5 min at 75°C, for 15 min at 37°C, and for 15 min at 25°C. The 5′ SR adapter (NEB) was resuspended in nuclease-free water (1:3) and denaturated by incubation for 2 min at 70°C. One microliter of denatured 5′ SR adapter (NEB), 1 μL of 10× ligation reaction buffer (NEB), and 2.5 μL of 5′ ligase enzyme mix (NEB) were added to samples. The samples were incubated for 60 min at 25°C. Eight microliters of First Strand Synthesis buffer (NEB), 1 μL of Murine RNase inhibitor (NEB), and 1 μL of ProtoScript II Reverse Transcriptase (NEB) were added to samples. The libraries were incubated for 60 min at 50°C and for 10 min at 75°C. The cDNA pool (40 μL) was purified using NucleoSpin Gel and PCR Clean up Columns for gel extraction and PCR clean up (Macherey-Nagel) according to PCR product purification protocol and eluted in 30 μL elution buffer. To the purified cDNA mixture we added 30 μL 2× Phusion Hot MasterMix (Thermo Fisher Scientific) and 1 μL of each 100 μM primer, universal primer, and barcoded primer. The following PCR was performed: initial denaturation and activation 1 min at 98°C, cycle denaturation 10 sec at 98°C, annealing 20 sec at 62°C, extension 5 sec at 70°C, and a final extension 5 min at 72°C. Nineteen cycles were performed. PCR product (80 μL) was purified in same manner as the cDNA pool and eluted in 25 μL elution buffer. Small PCR fragments were removed by adding 28 μL AMPureXP beads (Beckman), mixing, and incubating 10 min at room temperature. Beads were captured by magnet and the supernatant was removed. The pellet was resuspended in 30 μL nuclease-free water, placed back on the magnet, and the clear supernatant, ready sRNA library, was transferred to a tube. The library was quantified by KAPA SYBR FAST qPCR kit (Roche) according to instructions. The sRNA library was sequenced using an Illumina MiSeq (Illumina) instrument. The detailed protocol, and oligo and primer sequences are provided in Supplemental Methods and Supplemental Table S7, respectively.