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 the 4N Protocol A of Giraldez et al. (Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling, Nature biotechnology, 2018). For 3 4N adapter ligation, 1.5 l adapter mix (111 nM 3 4N adapter and 5.5% PEG 8000 [NEB]) were added and the samples were incubated at 70C for 2 min and immediately placed back on ice. Then, 2.5 l of the ligation master mix (200 U T4 RNA ligase 2 truncated KQ, 1x T4 RNA ligase buffer [both NEB], and 8 U recombinant RNase inhibitor [Clontech Takara]) were added and the samples were incubated at 25C for 2 h. The single stranded DNA binding protein (Promega) was diluted to 2 g/l using 1X T4 RNA ligase buffer (NEB), 0.5 l were added to every sample and incubation at 25C for 10 min was performed. After that, 25 U 5 deadenylase and 15 U RecJF (both NEB) were added sequentially and incubation at 30C for 1 h and 37C for 1 h respectively was performed. Next, the ligation of the 5 4N adapter was conducted: First, the 5 4N adapter was heated to 70C for 2 min, then 2 l of master mix (80 nM 4N 5 adapter, 400 M ATP [Thermo Fisher], and 10 U T4 RNA ligase 1 ssRNA [NEB]) were pipetted to every sample, and finally incubation was performed at 25C for 1 h. For reverse transcription, 6.5 l master mix (53 nM reverse transcription primer, 0.26 mM dNTPs [Roche], 0.8X first strand buffer, 5.26 mM DTT, and 200 U Super Script III reverse transcriptase [all Thermo Fisher]) were added and the samples were incubated at 55C for 1 h, followed by 37C for 15 min. Next, the cDNA was amplified using 30 l of the following PCR master mix: 2 M RP1 primer and 1X NEBNext Ultra II Q5 mastermix (NEB). Additionally, 2 M of an individual indexing primer were added to every sample. The following cycler program was applied:  98C for 30 s, 20 cycles of 98C for 10 s, 60C for 30 s, 65C for 35 s, and a final incubation at 65C for 2 min. The Ampure XP bead size selection was performed as described for the SB protocol. The 3 adapter was exchanged by the 3 CleanTag adapter and the 5 adapter was exchanged by an adapter with five 3 nucleotides complimentary to the 3 adapter and the UMI was shortened to 6 nt.