GSM5584792: S04: smxl5_GFP-; Arabidopsis thaliana; ATAC-seq
Approximately 1 g of Arabidopsis seedlings were collected in a 60 mm petri dish and thoroughly chopped using a razor blade for five minutes in nucleus isolation buffer (1x NIB Sigma-Aldrich, #CELLYTPN1) supplemented with 10µg/ml Hoechst 33342 (Sigma-Aldrich, #B2261) as the final concentration. Next, the samples were incubated 15 minutes in a dark cold room while gently shaking. After the incubation, petri dishes were washed with additional 1 ml of NIB to properly clean the plate. Using wide-bored pipet tips, the nucleus suspension was applied to a 50 µm nylon-mesh filter (Sysmex, #04-004-2327) mounted into a 30 µm nylon-mesh filter (Sysmex, #04-004-2326) at the top, both inserted into 2 ml Protein LoBind tubes (Eppendorf #0030108132). The filtered material was passed through a 5 ml Round Bottom Polystyrene FACS tube (Corning, #352235). 15,000 nuclei for each sample were then sorted into a DNA LoBind tubes (Eppendorf) with 300 µL collection buffer (15 mM TRIS-HCl ph 7.5, 20 mM NaCl, 80 mM KCl, 0,1 % Triton) by a BD FACSAriaTM IIIu cell sorter using a 100 µm sort nozzle and a 30kHz drop drive frequency according to their GFP signal levels. The gate for GFP + nuclei was set using a WT Col as reference. Next, samples were centrifuged at 3000 g for 10 minutes at 4 degrees. The supernatant was partially removed and the nuclei resuspended in 300 µL Tris-Mg buffer (Tris HCl pH8 10mM, MgCl2 5mM). A second washing step was performed using centrifugation at 1000 g for 10 minutes at 4 degrees and the supernatant was removed completely. For tagmentation, the TDE1 (Nextera Tn5 Transposase) Tagment DNA Enzyme (Illumina kit #20034198) was immediately applied to the samples by resuspending the nuclei in the transposition reaction mix and the suspension was incubated for 30 minutes at 37 degrees while gently shaking. The samples were then purified (NEB, Monarch Nucleic Acid Purification Kit #20034198), and 12 µl of transposed DNA was amplified by PCR (8 µl H2O, 2.5 µl 25µM Custom Nextera PCR primer 1 (Buenrostro et al., 2013), 2.5 µl 25 µM Custom Nextera PCR Primer 2 and 25 µl NEB Next High Fidelity 2x PCR Master Mix(NEB, #M0541) with 1 cycle of (72°C for 5 min, 98°C for 30 sec) and 16 cycles of (98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min). Library was cleaned up using the Coulter Agencourt AMPure XP kit (Beckman-Coulter, #10136224) applying a 1:1 vol:vol (PCR product:beads) and eluted in 15 µl 15 mM Tris.