GSM3611368: LacZ_10hpf_4; Ciona robusta; ATAC-seq
Nulcei were isolated from FACSorted cells and incubated in the tagmentation reaction. Tagmented-chromatin fragmented were PCR amplified and purified. The quality of ATAC-seq libraries was assessed by using 2100 Bioanalyzer (Agilent Technologies). ATAC-seq was performed with minor modifications to the published protocol (Buenrostro et al., 2013). 3000-5000 cells obtained by FACS were centrifuged at 800 x g for 4 min at 4 °C for each sample. Cells were resuspended in 5 µL of cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 nM NaCL, 3 nM MgCl2, 0,1% v/v Igepal CA-360 [Sigma-Aldrich] and incubated on ice for 5 min. After centrifugation of the cells at 500 x g for 10 min at 4 °C, the supernatant was discarded.  The tagmentation reaction was performed at 37 °C for 30 min using Nextera Sample Preparation kit (Illumina). After tagmentation, transposed DNA fragments were amplified using the following PCR condition: 1 cycle of 72°C for 5 min and 98°C for 30 s, followed by 12 to 14 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Amplified libraries were purified using PCR purification MinElute kit (Qiagen), and the quality of of the purified library was assessed by using 2100 Bioanalyzer (Agilent Technologies) and a High Sensitivity DNA analysis Kit (Agilent Technologies) to confirm a period pattern in the size of the amplified DNA. After size selection by using Agencourt Ampure XP beads (Beckman Coulter, Cat#A633880) with a bead-to-sample ratio of 1.8:1, the libraries were sequenced as paired-end 50 bp by using the HiSeq 2500 platform (Illumina) according to the manufacturer's instructions. An input control was also generated by using 10 ng of C. robusta genomic DNA.
GEO Accession
GSM3611368