design: The lung cancer cell line A549 (ATCC CCL-185) was cultivated in DMEM medium supplemented with 10% fetal bovine serum, 100 g/ml PenStrep, and 1x GlutaMAX. Adherent cell lines were passaged at 80% confluence using 1x Trypsin (PAN-Biotech) every 3-4 days. Non-adherent cells were splited 1:3 twice a week. Cells of passage 3-12 were washed with PBS (Life Technologies), centrifuged down (300 x g for 5 min), resuspended in PBS, and placed on AdcellTM diagnostic slides (Thermo Fisher Scientific). Single cells were isolated under the microscope in 1 l PBS using a micromanipulator (Patchman NP2) with pump (CellTram, both Eppendorf) and placed into 2l of lysis buffer (0.2% Triton X-100 [Sigma-Aldrich] and 4 U recombinant RNase inhibitor [Clontech Takara]). Samples were stored at -80C for up to six months. All pipetting steps were conducted on ice; master mixes were added to the edge of the PCR tube and centrifuged down unless otherwise stated. After thawing isolated single cells, 2 l of a 5 M 5.8S rRNA blocking oligonucleotide were added to every sample. Next, an incubation at 72C for 20 min, followed by cooling on ice, was performed. The SBN protocol (Hagemann-Jensen et al., Small-seq for single-cell small-RNA sequencing, Nature protocols, 2018) is an optimized version of the SB protocol. Three l of the 3 adapter ligation master mix (2 M 3 adapter, 8% PEG 8000, 50 U T4 RNA ligase 2 truncated KQ, 0.8x T4 RNA ligase buffer [all NEB], and 4 U recombinant RNase inhibitor [Clontech Takara]) were added, the samples were incubated at 30C for 6 h and then at 4C for 10 h. Removal of the unligated 3 adapter was performed using 2 l the following master mix: 5 M reverse transcription primer, 2.5 U Lambda Exonuclease and 25 U 5-Deadenylase (both NEB). The samples were incubated at 30C, followed by 37C for 15 min both. Then, the 5 adapter was ligated using a 2 l master mix: 1 M 5 adapter, 0.67 mM Tris-buffered ATP (Thermo Fisher), 13.5 U T4 RNA ligase 1 ssRNA and 0.25x T4 RNA ligase buffer (both NEB). An incubation for 1 h at 37C was performed. For reverse transcription, 5 l master mix (1.3x Taq DNA polymerase buffer, 0.5 mM dNTPs [both Roche], 8 mM DTT, 100 U Super Script II reverse transcriptase [both Thermo Fisher], and 4 U recombinant RNase inhibitor [Clontech Takara]) were added and the samples were incubated at 42C for 1 h, followed by 70C for 15 min. Next, the cDNA was amplified using 13 l of the following PCR master mix: 1 M RP1 primer, 0.15 mM dNTPS (Roche), 1 U Phusion Hot Start II DNA polymerase, and 1x Phusion buffer (both Thermo Fisher) and the cycler program:  98C for 30 s, 13 cycles of 98C for 10 s, 60C for 30 s, 72C for 30 s, and a final incubation at 72C for 5 min. In a second PCR amplification with a total volume of 25 l (800 nM RP1 primer, 0.2 mM dNTPs [Roche], 0.5 U Phusion Hot Start II DNA polymerase, and 1x Phusion buffer [both Thermo Fisher]) 1 ul of the first PCR was used as template and 2 M sample specific indexing primer were added. A similar cycler program as for the first PCR was used, but the annealing temperature was 67C. The Ampure XP bead size selection was performed as described for the SB protocol. The 3 and 5 adapters were replaced by the adapters of the modified CleanTag protocol.