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Sample GSM7880311

Query DataSets for GSM7880311

Status

Public on Nov 02, 2024

Title

RNAseq-L1-Spt6KD-rep2

Sample type

SRA

Source name

larva

Organism

Drosophila melanogaster

Characteristics

tissue: larva
time: L1
chip antibody: none
treatment: Spt6-RNAi

Extracted molecule

polyA RNA

Extraction protocol

cut&tag：Cells were immobilized on coA beads, antibodies were bound in antibody buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors, 0.05% digitonin, 0.01% NP-40, 2 mM EDTA), and activate Tn5-PA transposase activity in tagmentation buffer (10 mM MgCl2, 20 mM HEPES at pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors, 0.05% digitonin) , and cells were lysed to extract library fragments by add 1.5 μl 0.5 M EDTA, 0.5 μl 10% SDS and 1 μl 20 mg/mL proteinase K (sigma).
pol2 chip-seq：Cells were fixed with 1% paraformaldehyde for 10 min, and the chromosomes were fragmented using an ultrasonic crusher after lysis of the cells.
RNA-seq: RNA was extracted using trizol. 200 ng of total RNA was used to construct sequencing libraries.
cut&tag: 1×Agencourt AMPure XP beads (Beckman Coulter, A63881) were used to purify the tagementated DNA and eluted in 10 μl 0.1% Tween-20. The eluent was mixed with 10 U Bst 2.0 WarmStart DNA polymerase (NEB, M0538) in 1× Q5 polymerase reaction buffer, incubated at 65 °C for 30 minutes and stopped by incubation at 80 °C for 20 minutes. The universal i5 primer, barcoded i7 primer and Phanta EVO Super-Fidelity DNA Polymerase (Vazyme, P503) were added to amplify the R-loop CUT&Tag library. The libraries were size-selected with 0.56-0.85* Sera-Mag carboxylate-modified magnetic beads and eluted in 20 ul RNase-free water.
pol2 chip-seq: 5ng DNA was used for dsDNA library construction according to manufacturer’s instructions (Rapid Plus DNA Lib Prep Kit for Illumina V2, ABclonal, RK20255). The library was amplified for 13 cycles on the thermocycler.
RNA-seq: RNA libraries for Rna-Seq was prepared using MGIEasy RNA library Prep kit following manufacturer‘s protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-T7

Data processing

Reads trimmed by trim_galore were mapped to genome using bowtie2 (Cut&Tag and ChIP) or STAR (RNA-seq)
Redundant reads were deduplicated by picard
Peaks determintion was performed through Macs2 callpeak command. Deeptools bamCoverage converted BAM files into bigwig files
IDR was used to combine peaks of different replicates
FeatureCounts counted the read number on gene level for each sample
Assembly: dm6, Ecoli
Supplementary files format and content: Cut&Tag and ChIP-seq bigwig file and narrowPeak file
Supplementary files format and content: gene read count for RNA-seq

Submission date

Nov 02, 2023

Last update date

Nov 02, 2024

Contact name

Shaobo Liang

Organization name

Wuhan University

Street address

BaYi Road, LUOJIA HILL

City

Wuhan