design: MCF7 cells (ATCC HTB-22) were cultivated in RPMI medium supplemented with 10% fetal bovine serum, 100 g/ml PenStrep, 0.01 mg/ml human recombinant insulin (all PAN-Biotech) and 1x GlutaMAX (Life Technologies). 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. For the spike-in experiments, 1 l of the miRXplore Universal reference (Miltenyi) diluted to 0.96 pg/l was added to every sample (i.e. 10 fg of every spike-in miRNA). In order to mask 5.8S rRNA, 1 l of a 10 M blocking oligonucleotide (Metabion;) was added, the samples were incubated at 72C for 20 min, and immediately cooled on ice. 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.