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Sample GSM4476419 Query DataSets for GSM4476419
Status Public on Oct 28, 2020
Title SN_spk_rep1
Sample type SRA
 
Source name Oocyte
Organism Mus musculus
Characteristics tissue: Oocyte
genotype: wild-type
developmental stage: Full-grown oocyte SN
antibody: Pol2(Active Motif 102660)
Extracted molecule genomic DNA
Extraction protocol 8-week-old female mice were intraperitoneally injected with PMSG. Oocytes were harvested in M2 medium 48 h after PMSG. To distinguish SN from NSN oocytes without staining DNA, a previously established method was used (Inoue et al., 2007), as SN oocytes show a perivitelline space (a space between zona pellucida and the surface of the oocyte).
Antibody and pA-Tn5 (Vazyme Biotech, S603) or pG-Tn5 (Vazyme Biotech, S602) complex was prepared 20min ahead of sample lysis step. Add 1ul fully dissolved 5% digitonin into 1ml Buffer1 (10 mM Tris-HCl pH=7.4, 150mM NaCl, 0.5mM NaCl, 1x EDTA-free Roche complete protease inhitibor.) to prepare DB-1. Add 7μl DB-1, 0.5ul pA/G-Tn5 (choosing pA or pG depends on antibody species and affinity, we used pG-Tn5 for Pol II and pA-Tn5 for H3K4me3 and H3K27me3), 0.5ug antibody into 200ul low-binding tube and mix by vortex. Incubate at 4°C for 30min. Briefly, for Stacc-seq without wash step, fresh cells or embryos were prepared and put into 200ul low-binding tube (total volume with buffer should be less than 1μl). Add 6ul DB-1 into the sample and vortex. Incubate at 4°C for 10min, vortex each 2.5min for 3 times. Add 35μl DB-1 into the resulting samples, then add pA/G-Tn5 mixture and 12.5μl pre-warmed (37°C) 5x TTBL (Vazyme Biotech, TD502). Incubate in Thermo mixer at 37 °C for 30 min at 400 rpm. DNA was purified by phenol-chloroform with 40ng carrier RNA and 2ul spike-in DNA followed by ethanol precipitation 30min at -80°C or overnight at -20°C. PCR was performed to amplify the library for 16 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30s; and thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; followed by 72 °C 5 min. After the PCR reaction, libraries were purified with the 0.4x-1.7x AMPure (Beckman) beads size selection and were subjected to next generation sequencing. For Stacc-seq with wash step, fresh cells or embryos were suspended in 50ul Buffer1 into 200ul low-binding tube. 10ul Concanavalin-coated magnetic beads (Polyscience, 86057) were washed by 200 ul Buffer2 (10 mM Tris-HCl pH=7.4, 10mM KCl, 1mM CaCl2, 1mM MnCl2) once and resuspended in 10ul Buffer2. Add beads to samples and incubate for 10mins at room temperature. Place the samples on magnet stand and remove the supernatant and then wash beads with 200ul Buffer1 twice. Add pA/G-Tn5 mixture and DB-1 to 50ul to samples and resuspend the beads. Place tube on 4°C Thermo mixer at 400 rpm for 2h. Place the tube on magnet and remove the supernatant. Wash beads twice with 200ul DB-1. Resuspend the beads with 50ul DB-1 and add 12.5ul pre-warmed (37°C) 5x TTBL (Vazyme Biotech, TD502). Incubate in Thermo mixer at 37 °C for 30 min at 400 rpm. DNA was directly purified without removing beads by phenol-chloroform with 40ng carrier RNA and 2ul spike-in DNA followed by ethanol precipitation 30min at -80°C or overnight at -20°C. PCR was performed to amplify the library for 16 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30s; and thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; followed by 72 °C 5 min. After the PCR reaction, libraries were purified with the 0.4x-1.17x AMPure (Beckman) beads size selection and were subjected to next generation sequencing.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model HiSeq X Ten
 
Data processing Library strategy: Stacc-seq
Basecalls performed using CASAVA version 1.8
Stacc-seq reads were aligned to the mm9 genome using bowtie 2.2.2, replicates of same stage were pooled together and rpkm in wiggle files were counted by the number of reads falling into 114bp bin in the genome
Genome_build: mm9
 
Submission date Apr 14, 2020
Last update date Oct 29, 2020
Contact name Bofeng Liu
E-mail(s) lbf12thu@gmail.com
Organization name Tsinghua University
Department School of Life Science
Lab Xie Wei Lab
Street address Haidian District
City Beijing
ZIP/Postal code 100084
Country China
 
Platform ID GPL21273
Series (1)
GSE135457 The landscape of RNA Pol II binding reveals a step-wise transition during ZGA
Relations
BioSample SAMN14598138
SRA SRX8115742

Supplementary file Size Download File type/resource
GSM4476419_SN_RP_spk_rep1.bedGraph.gz 13.9 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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