GSM6340517: Keratinocytes_3; Homo sapiens; OTHER
A healthy whole human skin biopsy was obtained from the ilioinguinal region of a 62 y/o male donor of fair-skin type. The sample was immersed in MACS Tissue Storage solution (Miltenyi Biotec) immediately after resection and was kept on ice until further processing. The single-cell suspension from the epidermis was obtained using the Epidermis Dissociation Kit for human material (Miltenyi Biotec), and following the manufacturer´s instructions. We then followed the previously published sci-MET protocol (Mulqueen et al., 2018), with important modifications. Hence, cells were then fixed by incubation with 1.5% formaldehyde (without methanol) in 1 mL of PBS for 10 minutes, with gentle shaking. The reaction was stopped by adding 80 µL of 2.5 M glycine followed by 5 minutes incubation on ice. The sample was centrifuged at 550 g for 10 minutes at 4°C to recover fixed cells. Nuclear isolation was performed using the lysis buffer recipe described in (Wysocka et al., 2001) and as described for the single-cell multi-omics dataset. Recovered nuclei were subsequently subjected to nucleosome depletion. To do so, fixed nuclei were resuspended in 800 µl of 1X NEBuffer 2.1 (New England Biolabs) supplemented with 0.3% SDS, and incubated for 30 minutes at 42°C with intense shaking. Then, 200 µL of 10% Triton X-100 were added to the sample, which was incubated for 30 minutes at 42°C with intense shaking. Nuclei were then centrifuged at 500 g for 5 minutes at 4°C and filtered through a 40-µm cell strainer. The nuclear stain TO-PRO™-3 Iodide (1:10,000; Invitrogen) was then added, and we subsequently proceeded to fluorescence-activated nuclei sorting (FANS). In a first sorting step, we sorted 1,000 nuclei/well into a 96-well plate containing 5 µL of 2X Tagmentation Buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 140 µM PitStop 2 (Sigma-Aldrich), 20% DMF) and 5 µL of nuclear isolation buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630 (Sigma-Aldrich), 1X protease inhibitors cocktail). Next, 4 µL of a uniquely indexed transposome were added to each well, and the plate was incubated at 55°C for 30 minutes in a thermocycler for nuclear tagmentation. Importantly, transposomes were built by loading 96 uniquely barcoded cytosine-depleted oligonucleotides (Mulqueen et al., 2018) onto a homemade Tn5 transposase obtained from the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany). See Supplementary Methods for transposome assembly and activity test description. After tagmentation, nuclei from the 96 wells were pooled, mixed, and filtered through a 40-µm cell strainer again. Fresh TO-PRO™-3 Iodide (1:10,000; Invitrogen) was added for the second sorting step.  In this second sorting, we sorted 22 nuclei/well in a full 96-well plate containing 5 µL of Zymo Digestion Reagent (2.5 μL of Zymo M-Digestion Buffer, 2.25 μL of nuclease-free H2O, and 0.25 μL of Proteinase K), following the manufacturer's instructions of the EZ-96 DNA Methylation-Direct MagPrep Kit (Zymo Research).  Only 10 nuclei were sorted in three wells, where 35 pg of a barcoded-lambda DNA were added as spike-in controls. The plate was incubated for 4 hours at 50°C for nuclear digestion. Bisulfite conversion was then performed by adding 32.5 µL of Zymo CT Conversion Reagent in each well and incubating the plate according to the manufacturer´s instructions (98°C for 8 minutes and 64°C for 3.5 hours). After conversion, bisulfite-converted DNA was desulphonated, washed and recovered following the manufacturer´s instructions. In brief, 5 µL of Zymo M-Binding Beads diluted in 150 µl of Zymo M-Binding Buffer were added to each well, and the plate was incubated for 5 minutes at room temperature for allowing the DNA to attach to the beads. Beads were then washed with 80% ethanol, resuspended in 50 µL of Zymo M-Desulphonation Buffer and incubated at room temperature for 15 minutes. Beads were then washed again in 80% ethanol, and DNA was subsequently eluted in 25 µL of Zymo M-Elution Buffer. Eluted DNA was transferred to a 96-well plate containing 16 µL of nuclease-free H2O, 5 µL of 10X NEBuffer 2.1 (New England Biolabs), 2 µL of 10 mM dNTP mix, and 2 µL of 9-nucleotide random primers(Mulqueen et al., 2018). DNA was subjected to four rounds of linear amplification in a thermocycler by incubating at 95ºC for 45 seconds to achieve denaturation. The plate was quickly placed on ice, and 10 U of Klenow (3'-5' exo-) polymerase (Biozym Scientific) were added per well. The plate was placed again in the thermocycler for incubation at 4ºC for 5 minutes, followed by incubation at 37ºC for 90 minutes. Importantly, the temperature was increased to 37ºC by 1ºC/15 seconds. In each amplification round, fresh reagents were added to each well (1.25 µL of 4X NEBuffer 2.1(New England Biolabs), 1 µL of 10 µM dNTP mix, and 1 µL of 9-nucleotide random primers(Mulqueen et al., 2018)) as well as 10 U of Klenow (3'-5' exo-) polymerase (Biozym Scientific). Amplified material was subsequently purified using AMPureXP beads (Beckman Coulter) at a 1.1X volume. DNA was attached to the beads, washed with 80% ethanol, and eluted in 21 µL of EB Buffer (Qiagen). Eluted DNA was added to a 96-well plate containing 25 µL of 2X KAPA HiFi HotStart ReadyMix (Roche), 2 µL of 10 µM i7 index PCR primer, and 2 µL of i5 index PCR primer. Index PCR reaction was performed in a thermocycler by incubating at 95ºC for 2 minutes, then performing 18 cycles at 94ºC for 80 seconds and, finally, incubating at 65ºC for 30 seconds and at 72ºC for 30 seconds. The number of cycles was determined by a qPCR reaction performed with additional wells and could be up to 21 cycles. Libraries were then pooled to achieve a particular cell number and purified using a double-sided size selection step with AMPureXP Beads (0.6X-0.8X; Beckman Coulter) . The library was subsequently quantified with Qubit dsDNA HS Assay Kit (Invitrogen), and fragment size was assessed using D5000 or D1000 ScreenTapes (Agilent Technologies). Paired-end (150 bp) sequencing (100 cycles) was finally performed with a NextSeq550 High-Output (Illumina) system following a custom-made recipe (Read 1: 100 imaged cycles; Index Read 1: 10 imaged cycles; Index Read 2: 11 imaged cycles, 16 dark cycles and 10 imaged cycles). Sci-MET