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. After thawing isolated single MCF7 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. To produce single cell equivalents, the volume (5 l each) of all reactions was pooled, mixed thoroughly, and redistributed into fresh 0.2 ml PCR tubes. The protocol was adapted from Shore et al. (Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation, PLOS ONE, 2016). An 8 nt unique molecular identifier was introduced into the published 5 adapter analogously to the Sandberg protocol. For 3 CL adapter ligation, 6 l master mix (26 nM 3 CL adapter [Biomers], 13% PEG 8000, 200 U T4 RNA ligase 2 truncated KQ, 0.8x T4 RNA ligase buffer [all NEB], and 40 U recombinant RNase inhibitor [Clontech Takara]) were added to the samples and incubation was performed at 28C for 1 h and 65C for 20 min. Next, the ligation of the 5 CL adapter was conducted by adding 4.7 l master mix: 110 nM CL 5 adapter, 1.8 mM ATP, 10 U T4 RNA ligase, 0.45x T4 RNA ligase buffer (all Thermo Fisher) and 40 U recombinant RNase inhibitor (Clontech Takara). The same incubation conditions as for the 3 CL adapter ligation were used. Before reverse transcription, 18 nM reverse transcription primer were added, the samples were incubated at 70C for 2 min, and immediately placed back on ice. After that, 6.9 l of the reverse transcription master mix (7.65 mM DTT, 1x first strand buffer, 200 U Super Script II reverse transcriptase [all Thermo Fisher], 0.38 mM dNTPs [Roche], and 40 U recombinant RNase inhibitor [Clontech Takara]) were added and the samples were incubated at 42C for 1 h. Next, the cDNA was amplified using 17 l of the following PCR master mix: 2 M RP1 primer, 0.2 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 (200 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.