GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2283711

Query DataSets for GSM2283711

Status

Public on Aug 01, 2021

Title

RNAPII-RICh-PET_Rep1

Sample type

SRA

Source name

Organism

Drosophila melanogaster

Characteristics

cell type: S2
factor: RNAPII

Treatment protocol

Drosophila S2 cells were permeabilized with DPBS containing 0.015% Triton X-100 for 5 min at room temperature, then treated with or without RNaseA at a series of concentration at 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml respectively for 10min, then washing with DPBS.

Growth protocol

S2 was cultured following manufacture’s instruction.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin was obtained and subjected to fragmentation with an average length of 500 bp by sonication. The anti-RNAPII monoclonal antibody 8WG16 (Biolegend, 920102) was used to enrich RNAPII-bound chromatin fragments, respectively.
The DNA fragments in the streptavidin beads-bound chromatin for Standard RICh-PET or antibody beads-bound chromatin for RNAPII RICh-PET were end-repaired using T4 polymerase (Promega) in mixture (70 µl 10× T4 polymerase buffer, 7 µl 10mM dNTP, 3.5 µl RI, 612.3 µl Nuclease-free water, with 7.2 µl of T4 polymerase), washed with RICh-PET Wash Buffer (10 mM Tris.HCl pH 7.0, 10 mM Tris.HCl pH 8.0, 10 mM EDTA, 50 mM NaCl) three times. The RNA fragments were linked to RNA linker for first-strand cDNA synthesis using the Superscript III First Strand Synthesis System (Thermo Fisher Scientific). In more details, 1 µg of biotinylated and barcoded RNA linker R1 (tube 1) and R2 (tube 2) containing a flanking MmeI site were added to two tubes containing cDNA synthesis mixture (20 µl RNA liker, 20 µl dNTP, 80 µl 5× RT buffer, 20 µl 0.1 M DTT, 20 µl RI, 220 µl Nuclease-free water) and incubated at 65 °C for 5 min (Alternative, incubated at 42 °C for 5 min when performing RNAPII RICh-PET), then placed on ice for at least 1 min, and added 20 µl superScriptIII RT, and incubated for 10 min at 25 °C on Intelli-Mixer (Program F1, 10rpm), followed by 30 min at 50 °C (Alternative, 1 hour at 37 °C when performing RNAPII RICh-PET). The RNA linker ligated chromatin was washed with Wash Buffer three times, then incubated with 1 µg of DNA linker D1 (tube 1) and D2 (tube 2) respectively in the ligation mixture (15 µl DNA linker, 140 µl 5× T4 DNA ligase buffer with PEG, 3.5 µl RI, 536.5 µl Nuclease free water) by using 5 µl of T4 DNA ligase at 16 °C for overnight. The linker-added DNA fragments were washed with Wash Buffer three times and then phosphorylated with 14 µl of T4 polynucleotide kinase (NEB) in PNK master mix buffers (70 µl 10× T4 DNA ligase buffer, 3.5 µl RI in 612.5 µl Nuclease-free water). The samples from two tubes were washed with Wash Buffer three times and then were mixed and followed by proximity ligation with 34 µl of T4 DNA ligase in reaction buffer (1,000 µl 10× T4 DNA ligase buffer, 50 µl RI, 8,916 µl Nuclease-free water) overnight at 16 °C. After washing with Wash Buffer three times, the chromatin DNA fragments with linkers were subjected to second-strand cDNA synthesis with a Superscript Double-stranded cDNA Synthesis Kit (Thermo Fisher Scientific) and chromatin mixing with second-strand cDNA mixture (333 µl Nuclease-free water, 90 µl 5× second-strand reaction buffer, 9 µl 10 mM dNTP mix, 3 µl E.coli DNA ligase, 12 µl E.coli DNA Polymerase I, 3 µl E.coli RNase H) were incubated at 16 °C for 2 hours; then 6 µl of T4 DNA polymerase were added and continued to be incubated at 16 °C for 5 min. Crosslinks in the DNA/RNA/protein complexes were then reversed by incubation at 65 °C overnight with 0.5% SDS (Thermo Fisher Scientific) and proteinase K (Thermo Fisher Scientific), and the cDNA-DNA fragments were purified by phenol/chloroform isopropanol precipitation. The purified cDNA-DNA was then digested by 1 µl of MmeI (NEB) in buffer (5 µl 10× NE Buffer 4, 5 µl Half linker non-Biotinylated to quench excess MmeI, 5 µl 10× SAM) for 2 hours at 37 °C to release the [cDNA tag]-[RNA linker]-[DNA linker]-[DNA tag] Paired-End-Tag structure. The biotinylated PETs were then immobilized on streptavidin-conjugated magnetic Dynabeads (Thermo Fisher Scientific, 11206D-10ml) in 50 µl of 2× B&W buffer (10 mM Tris-HCl pH 7.5, 1mM EDTA, 1 M NaCl), and rotated at room temperature for 45 min. The ends of each PET structure were then ligated to a sequencing adaptor by 1 µl of T4 DNA ligase (Thermo Fisher Scientific, EL0013) in Adaptor ligation buffer (4 µl Adaptor A, 4 µl Adaptor B, 5 µl 10× T4 DNA ligase buffer, 36 µl Nuclease-free water) at 16 °C for overnight with rotation. Then the beads were washed three times with 1× B&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). Nick translation was performed by adding 4 µl of E.coli DNA polymerase I solution mixture (38.5 µl Nuclease free water, 10× NEBuffer 2, 2.5 µl 10 mM dNTP) and incubated at room temperature for 2 hours with rotation on the Intelli-Mixer (F8, 30rpm, U = 50, u = 60). Then 10-16 rounds of PCR reactions were performed to amplify the PETs, and the resultant RICh-PET libraries were subjected for sequencing by HiSeq2500 with paired-end reads of 36 bp.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

nuclear chromatin associated RNA
Replicate 1

Data processing

Library strategy: RICh-PET
RICh-PET raw reads were filtered by linker sequence composition and the PET reads with specific ligation linkers (R1D1 or R2D2 linkers) were kept for downstream mapping and analyses. BatMis (Tennakoon et al., 2012) was used to map the retained PET sequences to human genome build dm3. The mapping results were then filtered based on very stringent criteria: 1) both ends of the PET sequence had to be uniquely mapped (allowing a maximum of two mismatches on each end); 2) PET sequences with both ends mapped to precisely the same locations were merged and considered as one read, i.e., redundant reads were removed. With the mapped PETs, linker barcodes were used to distinguish the tag sequences that originated from the RNA molecules (RNA-tags) from the target chromatin DNA-tags.
ChIRP-Seq bioinformatics analysis was performed as previously described (Quinn, J.J. et al. 2014). Raw fastq data were mapped to dm3 reference genome by BWA mem with default parameters and minimum seed length (-k) set to 18. Duplicate reads were removed by using the Picard toolkit (http://broadinstitute.github.io/picard/). Only uniquely mapped reads (mapping score ≥ 20) were kept for further analysis. ChIRP-Seq peaks were identified by using MACS (version 2.0.10) with configuration of --nomodel and --keep-dup set to all.
ATAC-Seq raw reads in fastq format were first aligned to Drosophila dm3 genome assembly with BWA mem. PCR redundancy was removed by using the Picard toolkit (http://broadinstitute.github.io/picard/). Sequences with alignment score ≥ 20 were retained for further analysis. For each ATAC-Seq library, ATAC-Seq peaks were identified by using MACS (version 2.0.10) with default parameters.
Data processing of ChIA-PET reads was performed as previously described in Li et al., 2012 with slight modifications.
Pair-end read (PET) sequences were scanned for the bridge linker sequence and only PETs with the bridge linker were used for downstream processing. After trimming the linkers, the sequences flanking the linker were mapped to the dm3 or hg19 using bwa-mem (Li and Durbin, 2010) and only uniquely aligned (MAPQ ≥ 30) PETs were retained. PCR duplicates were removed using the MarkDuplicates tool of the Picard Tools library (http://broadinstitute.github.io/picard/). Each PET was categorized as either a self-ligation PET (two ends of the same DNA fragment) or inter-ligation PET (two ends from two different DNA fragments in the same chromatin complex) by evaluating the genomic span between the two ends of a PET.
PETs with a genomic span less than 8 kb are classified as self-ligation PETs and are used as a proxy for ChIP fragments since they are derived in a manner analogous to ChIP-Seq mapping for protein binding sites. PETs with a genomic span greater than 8 kb are classified as inter-ligation PETs and represent the long-range interactions of interest. To accurately represent the frequency of interaction between two loci and to define the interacting regions, both ends of inter-ligation PETs were extended by 500 bp along the reference genome, and PETs overlapping at both ends (with extension) were clustered together as one PET cluster.
Genome_build: dm3 and hg19
Supplementary_files_format_and_content: RICh-PET:Tab-delimited file documenting the genomic coodinates of DNA and RNA of each interaction PET with RNA strand information; ChIA-PET: Tab-delimited file documenting the genomic coodinates and frequency of each interaction PET clusters.

Submission date

Aug 17, 2016

Last update date

Aug 01, 2021

Contact name

Zhongyuan Tian

E-mail(s)

simon.tian@jax.org

Phone

860-837-2358

Organization name

The Jackson Laboratory for Genomic Medicine