GSM3265768: GFP+_rep1; Arabidopsis thaliana; ATAC-seq
Inflorescence apices of approximately 700-900 DRNL::erGFP ap1cal plants were collected  and FACS was performed as described in Frerichs et al., 2016. After separation of GFP-positive (GFP+) and GFP-negative (GFP–) cells with a FACS Vantage SE (Becton Dickinson) sorter for a maximum period of 1 h (36,000 cells s-1 flow rate; 100 µm aperture), the protoplasts were directly collected into 10 mL FACS-medium. The number of collected GFP+ protoplasts typically varied between 95,000 to 300,000 per experiment, for comparison approximately 300,000 GFP− protoplasts were collected as control. Protoplasts treatment after FACS separation followed the protocol of (Buenrostro et al., 2013) with some adaptations. Protoplasts in FACS medium were collected by centrifugation (500 rpm, 10 min, 4 °C), washed once in 500 µL FACS-medium and pelleted by centrifugation before cells were lysed in 10 mM TrisHCl pH 7,4, 10 mM NaCl, 3 mM MgCl2, 0,1 % IGEPAL CA-630 for 30 min on ice. After centrifugation (1000 rpm, 4 °C), the nuclei were washed twice successively for 10 min or 2 min in lysis buffer before being resuspended in 15 µl nuclease-free H2O. 25 µl 2x TD Tagment DNA buffer and 10 µl TDE1 Tagment DNA Enzyme (TruSight One Kit) were added to a total reaction mix of 50 µl followed by an incubation at 37 °C for 30 min with gentle mixing every 5 min. Subsequently nuclear DNA was isolated with the MinElute PCR Purification Kit (28004, Qiagen, Hilden, Germany) and eluted in 10 µL elution buffer. For Input control samples, inflorescences (0.6 g) of DRNL::erGFP ap1cal plants were directly grinded in liquid nitrogen and the powder resuspended in 20 ml nuclei isolation buffer (NIB:10 mM NaPO4 pH 7, 100 mM NaCl, 1 M 2-methyl-2,4-pentanediol, 10 mM β-Mercaptoethanol, spplementation with SigmaFAST Protease Inhibitor (S8820)). Cell debris was removed by sieving (mesh 100 µm), the sieve was washed once with 5 ml NIB, nuclei were pelleted by centrifugation (1,000 g, 20 min, 4 °C). The pellet was resuspended and centrifuged 5-times with 5 ml NIB supplemented with 10 mM MgCl2, 0,5 % (v/v) Triton X-100 before purified nuclei were resuspended in sonification buffer (10 mM NaPO4 pH 7, 10 mM NaPO4 pH 7, 100 mM NaCl, 17 mM Sarkosyl, 10 mM EDTA, SigmaFAST Protease Inhibitor) and sonified in a Branson Sonifier II w-450 (80 % Duty Cycle, 7 Output Control) for 18 min at 4 °C to a fragment size of 200-500 bp. Chromatin was cleared twice by centrifugation at 21,000 g (4 °C) before genomic DNA was isolated using the Qiagen MinElute PCR Purification Kit (28004, Qiagen, Hilden, Germany) and eluted in 10 µl elution buffer. Libraries for the purified control DNA were constructed using Illumina's TruSeq DNA nano kit. For the ATAC-seq samples, purified tagmented DNA was amplified via a 12 or 15-cycle PCR program using reagents from the Illumina TruSight One Library Preparation Kit. This procedure adds index 1 (i7) and index 2 (i5) information needed for multiplexing, as well as common adapters (P5 and P7) required for cluster generation and sequencing. To remove unwanted products the final Library has been purified by using Beckman Coulter Ampure XP Beads. After purification and validation (2200 TapeStation; Agilent Technologies), libraries are quantified by using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems) and subsequently sequenced on an Illumina HiSeq4000 sequencing system and a paired end 2x75nt sequencing protocol.