GSM4432951: EMB76_ADF; Arabidopsis thaliana; OTHER
Frozen tissue was grinded in liquid nitrogen using mortar and pestle, and transferred into Nuclear Isolation Buffer 1 (10 mM HEPES pH=7.5, 0.4 M sucrose, 5 mM KCl, 5mM MgCl2, 5 mM EDTA, supplemented with 0.2 mM Spermine, 0.5 mM Spermidine and 0.6% Triton X-100) and incubated on a rotator at 4ºC for 30 min. The sample was then passed through 40µm mesh and centrifuged (2500g at 4°C). The pellet was resuspended with Nuclear Isolation Buffer 2 (10 mM HEPES pH=7.5, 0.25 M sucrose, 5 mM KCl, 5mM MgCl2 and 5 mM EDTA). The debris was further reduced by two rounds of gentle centrifugation at 300g at 4°C for 15 sec and collecting the supernatant. The supernatant was further centrifuged for 10 min, at 2000g at 4°C and the resulting nuclei pellet was re-suspended with NIB2. For crosslinking, the sample was incubated with 1% formaldehyde at room temperature for 10 min and neutralized with125mM glycine for 5 minutes at room temperature. 10^6 crosslinked nuclei were washed twice with Csp6I RE buffer (Thermo, #ER0211) and resuspended in 1.2X Csp6I RE 1.2X. SDS was added to a final concentration of 0.3% (v/v). After 30 minutes incubation at 65°C, triton X- 100 was added to final concentration of 1.8% and the sample was incubated for 1h in 37°C. 150 units of restriction enzyme Csp6I (Thermo, #ER0211) were added and the sample was incubated overnight at 37°C. Inactivation of Csp6I was achieved by incubating the samples at 65°C for 25 minutes. Ligation reaction was then done in 1.5ml of 1X ligase buffer with 25 units T4 ligase (Roche, #EL0011) and incubated over-night at 4°C followed by 30 minutes incubation at RT. To reverse the crosslinks the sample was supplemented with 50µg proteinase K (Invitrogene, #25530049) and incubated overnight at 65°C, followed by incubation with 150 µg RNAse (Thermo, EN0551) for 45 min at 37°C. The resulting proximity-ligated DNA was extracted by phenol-chloroform followed by ethanol precipitation and dissolved in 20 µl TE buffer. For generating DNA circles, DNA was then digested over-night with DpnII (NEB, #R0543T) at 37°C.  DNA was purified using Expin DNA clean up kit (GeneAll, 112-102) and DNA concentration was determined by Nanodrop 2000 (Thermo Scientific).Ligation junctions were amplified by inverse PCR with viewpoint primers and Phusion hot-start high fidelity Taq (Thermo, #F-549) and the following thermal cycles conditions: 30 sec at 98°C ; 25 cycles of 98°C for 10 sec, 60°C for 30 sec and 72°C for 2 min; 72°C for 5min. PCR products were purified on Expin columns. 30, 120, and twice 60 ng (in total four amplified libraries) DNA templates from SALK and ADF8p:NTF/ACT2p:BirA lines were amplified separately with primer-specific barcodes. For further varying the template amounts, the sequenced tags from these libraries were combined. Libraries for Illumina sequencing were prepared using NEBNext Ultra II system (NEB, #E7645, #E7103) and sequenced on a NextSeq platform (Illumina). For multiplexing several samples on the same sequencing lane, 4-letter barcodes were added to the primers used for inverse PCR. 4C-seq
GEO Accession
GSM4432951