GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5597392

Query DataSets for GSM5597392

Status

Public on Apr 13, 2022

Title

iMIP_rep2

Sample type

SRA

Source name

seedling

Organism

Oryza sativa Japonica Group

Characteristics

chip antibody: iMab

Growth protocol

Nipponbare (Japonica) rice seeds (Oryza. sativa L.) were pre-germinated at room temperature (RT) for three days. Germinated seeds were grown in a greenhouse at 28-30°C and a 14h/10h light-dark cycle. Two-week-old rice seedlings were cut into1-1.5 cm slices and cross-linked in HEPES buffer pH=8.0 (20mM HEPES, 1mM EDTA, 100mM NaCl and 1mM PMSF) with a final 1% of formaldehyde under vacuum for 10 min at RT. A final concentration of 0.125 M glycine was added for an additional 5min vacuum for quenching excess of formaldehyde. The cross-linked seedlings were ground to a fine powder using liquid nitrogen.

Extracted molecule

genomic DNA

Extraction protocol

The fine powder was used for nuclei preparation and genomic DNA extraction following the procedures as described previously
The genomic DNA was fragmented into sizes, ranging from 100-500 bp, using the water-based Biorupter (Diagnode), followed by DNA extraction and purification. Total 5μg fragmented genomic DNA was diluted in iM stabilization buffer (50mM Tris-AcOH, pH=5.5) for denaturing and re-association treatment using a PCR program as below: 95°C for 8 min，95°C for 30s (-0.5 °C /cycles, 129 cycles)，35°C hold on. The re-associated DNA was diluted with iM-IP incubation buffer (50mM Tris-AcOH, 1mM MgCl2, 130nM CaCl2, 1%BSA and Complete mini, pH=5.5), then incubated with 3μg iMab antibody (Ab01462-23.0, Kappa) for 4 h at 4℃. The antibody incubation reaction was incubated with 30 μl of washed protein G Dynalbeads (10004D, Invitrogen) for another 4 h at 4°C. The iMab-bound DNA was finally eluted with 200 μl elution buffer (0.1M NaHCO3 and 1% SDS) at 65°C for two times with 15 min each. Three biologically replicated iM-IPed DNA or Input/IgG-IPed control DNA was used for library preparation. All libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S). Libraries were finally quality controlled and sequenced using paired-end mode on Illumina NovaSeq platform.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Data processing

All clean reads in three biological replicates were aligned to the MSU v7.0 reference genome using BWA
SAMtools was used to exclude reads with mapping quality below 10
PCR duplicates were removed by using Picard
Aligned reads with at least length 50 were used for iM-IP peak calling by using MACS2 (version 2.1.1) (42)with parameters as below: callpeak -g 3.8e+8 -p 1e-4 -nomodel -f BAMPE
Supplementary_files_format_and_content: a bed file containing information of regular PiMFSs

Submission date

Sep 26, 2021

Last update date

Apr 13, 2022

Contact name

Xing Ma

E-mail(s)

2020101156@stu.njau.edu.cn

Organization name

Nanjing Agricultural University

Street address

No.1 Weigang, Nanjing, Jiangsu 210095, P. R. China