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| Status |
Public on Sep 23, 2022 |
| Title |
retina, P28, NRL, 2 |
| Sample type |
SRA |
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| Source name |
retina
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| Organism |
Mus musculus |
| Characteristics |
tissue: retina
age: postnatal day 28 (P28)
antibody: anti-NRL
replicate: 2
strain: C57BL/6J
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Retinas were dissected at P2, P4, P10, and P28 and cells were dissociated using dissociation solution (30 U/mL papain, HBSS pH 7.4, 1 mg/mL glucose, 10 mM Hepes, 100 U/mL DNase I, 5 µg/mL superoxide dismutase, 5 µg/mL catalase, 10 µg/mL D-alpha-tocopherol acetate, 1 mg/mL glucoscysteine-HCl, 5 µg/mL superoxide dismutase, 50 µg/mL gentamycin). Cells were pelleted at 200g for 5 min, washed and re-suspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM Spermidine) with Complete Protease Inhibitor tablet (MilliporeSigma, Burlington, MA, USA). Concanavalin A–coated beads (Polysciences, Warrington, PA, USA) were incubated with the cell suspension containing 250,000 cells for 10 min, washed to remove unbound cells, and incubated with NRL antibody (1:200) (Cat#ab-137193, Abcam, Cambridge, UK) in Antibody Buffer (20 mM EDTA and 0.02% digitonin in Wash Buffer) for overnight. Next day beads were washed with Wash Buffer containing 0.02% digitonin and incubated with 700 ng/ml pA-MNase for 1 hr at 4 °C. Beads were washed, re-suspended in 100 ul of Wash Buffer containing 0.02% digitonin and incubated with 2 mM CaCl2 at 0 °C for 30 min. Cleavage reaction was quenched by adding 100 ul 2X Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 25 μl RNase A, 0.05 mg glycogen and 10 pg heterologous spike-in DNA). DNA fragments were released from insoluble nuclear chromatin by incubation at 37°C for 10 min and extracted by spin column (Qiagen, Hilden, Germany).
Libraries were prepared with ThruPLEX DNA-seq kit (Takara Bio USA, Inc, Mountain View, USA).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
postnatal day 28 retina probed with anti-NRL anitbody replicate 2
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| Data processing |
Library strategy: CUT&RUN
Sequencing reads were trimmed using Trimmomatic v0.38 for library adapters, 6-base tail cropping, end-wise PHRED 20 quality score, and requiring a minimum length of 25 bases for further analysis.
Trimmed sequencing reads were aligned to a chimeric index using bowtie2 v2.3.5.1 with the following settings: --end-to-end --dovetail -I 10 -X 700 --very-sensitive --no-unal --no-mixed --no-discordant -q --phred33. A chimera bowtie2 index was created by concatenating genomic DNA fasta files of Mus musculus GRCm38, Saccharomyces cerevisiae S288C, and Escherichia coli ASM584v2 prior to running the bowtie2 index function.
Quality alignments were kept if MAPQ scores were greater than 20 using samtools v1.9 (Li, Handsaker et al. 2009). Duplicate aligned reads were removed using picard v2.20.8 (https://broadinstitute.github.io/picard/) MarkDuplicates algorithm. Peaks were identified using MACS2 v2.2.5 (Zhang, Liu et al. 2008). Called peaks overlapping ENCODE blacklist regions (Amemiya, Kundaje et al. 2019) were removed using Bedtools v2.29.0 (Quinlan and Hall 2010).
The Irreproducible Discovery Rate approach was used to determine consensus peaks at each timepoint using IDR v2.0.3 with an IDR threshold of 0.05.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: bigwig, peaks. peak_quantitation.tsv: represents the quantitation for the individual samples used in the IDR peak analysis at each timepoint (2 replicates x 4 timepoints; for P2, P4, and P28 replicates 3 and 4 were used, for P10 replicates 1 and 4 were used). Take the mean at each timepoint to get the final figure data.
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| Submission date |
Feb 24, 2022 |
| Last update date |
Sep 23, 2022 |
| Contact name |
Matthew J Brooks |
| E-mail(s) |
[email protected]
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| Phone |
301-443-4906
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| Organization name |
NIH
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| Department |
NEI
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| Lab |
NNRL
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| Street address |
6 Center Dr Bldg 6, Rm 303
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE197420 |
Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina [CUT&RUN] |
| GSE197421 |
Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina |
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| Relations |
| BioSample |
SAMN26235870 |
| SRA |
SRX14282901 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5917173_P28_NRL_2_bpm.bw |
72.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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