Single cell isolation from human organs: The tissue culture dishes to use for the single cell picking were first coated for a couple of minutes at room temperature with 0.5% bovine serum albumin (BSA, Life Technologies, Carlsbad, USA) in PBS and then filled with Dulbecco’s Modified Eagle Medium/F12 Nutrient mixture (DMEM/F12, Life Technologies, Paisley, UK). Human gonads and adrenals were placed individually in tissue culture dishes and the organs were mechanically disrupted with sharp needles so that single cells were released. Single cells were then manually picked under a stereo microscope (Zeiss, Sliedrecht, the Netherlands) using a pulled glass capillary in picking volumes of ±0.5µl medium and transferred to ice-cold PCR tubes containing 2.0µl lysis buffer (1.9µl 0.2% TritonX-100 in water + 0.1µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A) per tube), snap-frozen on dry ice and stored at -80°C.  Reverse transcription: Single-cell full-length cDNA libraries were prepared using the Smart-seq2 protocol (Picelli et al., 2013). Briefly, 2.1µl priming buffer mix (1µl 10mM dNTPs; 1µl 10µM Smarter oligo-dT primer was added to the cell lysate and incubated for 3 min at 72°C in a thermal cycler, and then put on ice. Reverse transcription (RT) was performed by the addition of 5.6µl Smart-Seq2 RT mix (0.5µl SuperScriptII (Invitrogen Cat. 18064-014); 2µl 5x SuperScriptII buffer; 0.5µl 100mM DTT; 2µl 5M betaine; 0.1µl 1mM MgCl2; 0.25µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1µl 100µM LNA strand switch primer; 0.15µl water, per reaction) and incubation (90 min 42°C; 10 “strand-switch” cycles of (2 min 50°C; 2 min 70°C); 4°C) in a thermal cycler.   cDNA amplification: cDNA amplification was performed by the addition of 15µl of Smart-Seq2 PCR mix (12.5µl 2x KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25µl 10µM ISPCR primers; 2.25µl water, per reaction) and incubation (3 min 98°C; 19 cycles of (20 s 98°C; 15s 67°C; 6 min 72°C); 5 min 70°C; 4°C) in a thermal cycler. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. A63882) according to the manufacturer’s protocol (No. PT5163-1). The purified cDNA for each cell was inspected on an Agilent 2100 Bioanalyzer to determine cDNA concentration and size distribution, using Agilent High Sensitivity DNA chips (Ref. 5067-4626).   Tagmentation and sequencing: Successful cDNA libraries were tagmented using the transposase Tn5 (Picelli et al., 2014). 1ng of cDNA in 5µl water was mixed with 15µl tagmentation mix (1µl of Tn5; 2µl 10x TAPS MgCl2 Tagmentation buffer; 5µl 40% PEG8000; 7µl water, per reaction) and incubated 8 min at 55°C in a thermal cycler. Tn5 was inactivated and released from the DNA by the addition of 5µl 0.2% SDS and 5 min incubation at room temperature. Sequence library amplification was performed using 5µl Nextera XT Index primers (Illumina, Ref. 15032356) and 15µl PCR mix (1µl KAPA HiFi DNA polymerase (KAPA Biosystems Ref. KK202); 10µl 5x KAPA HiFi buffer; 1.5 µl 10mM dNTPs; 2.5µl water, per reaction), and incubation (3 min 72°C; 30 s 72°C; 10 cycles of (10 s 72°C; 30 s 55°C; 30 s 72°C); 5 min 72°C; 4°C) in a thermal cycler. Sequencing libraries were purified using 1:1 volume of AMPure XP beads (Beckman Coulter, Ref. A63882) according to the manufacturer’s protocol (No. PT5163-1, version PR0X3693), inspected on an Agilent 2100 Bioanalyser, using Agilent High Sensitivity DNA chips (Ref. 5067-4626), and the DNA concentration was measured using a Qubit 2.0 Fluorometer (Invitrogen) with the Qubit dsDNA High Sensitivity Assay kit (Molecular Probes, Ref. Q32854). Pools of samples for multiplexing, with unique Illumina barcode for each cell, were prepared according to the Nextera XT DNA Sample Preparation Guide (No. 15031942 page 46, llumina). DNA sequencing was performed on an Illumina HiSeq2000 (43bp single-end) and on Illumina NextSeq500 (75bp single-end).
GEO Accession
GSM2090611