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 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 and 5 adapters were replaced by the adapters of the modified CleanTag protocol.