title: GSM4157878: E_C1_R1; Danio rerio; ATAC-seq
library_construction_protocol: The chorion was removed with pronase (0.3 mg/mL pronase) and the yolk was dissociated in 500 μL Ginzburg fish ringer deyolk buffer (55 mM NaCl, 1.8 mM KCl,1.25 mM NaHCO3) and the cell membrane was lysed in 50 μL lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA630). A 2X DNA tagmentation buffer (22 mM Tris-HCl pH 7.4, 10 mM MgCl2, 20% Dimethylformamide; pH 7.4) was prepared. The isolated nuclei were resuspended in 50 μL transposition reaction mixture (25 μL DNA tagmentation buffer, 1.25 μL Tn5 (Illumina, 15027865), 23.75 μL H2O). The samples were incubated on Thermomixer at 300 rpm, 37 °C for 30 minutes. The tagmented DNA was purified with the MinElute PCR Purification Kit (QIAGEN, cat. #28004) and eluted in 10 μL elution buffer. The Nextera Index Kit (Illumina, FC-121-1030) was used for the library preparation.Real-time PCR was performed using 1 μL of the transposed DNA and 9 μL qPCR master mix (1X NEB Next High-Fidelity PCR Master Mix (New England Labs, cat. #M0541), 1X SybrGreen, 0.25 nM indexed primers) to determine the number of PCR cycles needed for the library preparation. The temperature regime for the thermocycling was (1) 72 °C for 5 minutes, (2) 98 °C for 30 seconds, (3) 98 °C for 10 seconds, (4) 63 °C for 30 seconds, and (5) 72 °C for 30 seconds. Step 3 - 5 was repeated 20 times. The preferred number of amplification cycles for the libraries was decided to be 9-10 cycles.The remaining 9 μL of purified transposed DNA was PCR amplified with 41 μL PCR master mix (1X NEB Next High-Fidelity PCR Mastermix (New England Labs, cat #M0541), 5 μM common and specific index primers (Nextera Index Kit, Illumina). The temperature regime for the thermocycling was (1) 72 °C for 5 minutes, (2) 98 °C for 30 seconds, (3) 98 °C for 10 seconds, (4) 63 °C for 30 seconds, and (5) 72 °C for 60 seconds. The amplified library was purified using the MinElute PCR Purification Kit and eluted in 20 μL of elution buffer. The libraries were cleaned up on a 2% agarose gel (Xe dry gel system, Invitrogen). During blue light excitation, the <100 bp and >15 kb localizations were removed, and the DNA libraries of interest were extruded from the gel using Invitrogen PureLink Quick Gel Extraction Kit (Invitrogen, cat. #K2100).The libraries were pair-end sequenced with a read length of 150 bases and a read depth of 50 million raw reads. The sequencing was outsourced to Novogene, Hong Kong, China.
attributes.tag: GEO Accession
attributes.value: GSM4157878