Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Neural

Cell type

Retina

MeSH Description

The ten-layered nervous tissue membrane of the eye. It is continuous with the OPTIC NERVE and receives images of external objects and transmits visual impulses to the brain. Its outer surface is in contact with the CHOROID and the inner surface with the VITREOUS BODY. The outer-most layer is pigmented, whereas the inner nine layers are transparent.

Sample

source_name

Rods from WT mouse retina

strain background

C57BL6J/129

genotype/variation

LMOPC1-Cre; R26-LSL-CAG-Sun1-GFP-myc

age

8 to 11 weeks

Sex

male

tissue

retina

cell type

WT rod photoreceptors

chip antibody

none (input)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

The retina was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401). Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). ChIP-enriched and input DNA was end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles. RNA-seq: Mice retinas were rapidly dissected on ice, and either the INTACT method or flow cytometry was used to purify GFP+ nuclei. Isolated nuclei were resuspended in Buffer RLT for RNA purification using the RNeasy Micro kit (Qiagen 74004) with on-column DNase digestion following the standard kit protocol. RNA quality was measured by an Agilent Bioanalyzer. RNA-seq: For preparation of nuclei from the whole retina, the retina was rapidly dissected from 1 adult mouse on ice, homogenized, and nuclei were pelleted by centrifugation. Pelleted nuclei were resuspended in Buffer RLT for RNA purification using the RNeasy Micro kit (Qiagen 74004) with on-column DNase digestion following the standard kit protocol. RNA quality was measured by an Agilent Bioanalyzer. Bisulfite-Seq (MethylC-seq): Mice retinas were rapidly dissected on ice, and either the INTACT method or flow cytometry was used to purify GFP+ nuclei. Isolated nuclei were resuspended in PBS for DNA purification using the DNeasy Blood and Tissue kit (Qiagen 69504) following the standard kit protocol. ATAC-seq: Mice retinas were rapidly dissected on ice, and the INTACT method was used to purify GFP+ nuclei. ChIP-seq: Mice retinas were rapidly dissected on ice, and the INTACT method was used to purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401). RNA-seq: RNA (2-50ng) was converted to cDNA and amplified using Nugen Ovation RNA-seq System V2 (Nugen 7102). All RNA samples received a 1:10,000 dilution of ERCC RNA (Life Technologies 4456740). Amplified cDNA was fragmented, end-repaired, linker adapted, and sequenced for 50 cycles on an Illumina HiSeq 2500 instrument. Bisulfite-Seq (MethylC-seq): MethylC-seq libraries were constructed as previously described (Mo et al., 2015). Libraries were sequenced on an Illumina HiSeq 2000 up to 101 cycles. OTHER: ATAC-seq: Approximately 50,000 bead-bound nuclei were transposed in a 50uL volume of 1X TD buffer and 2.5uL Tn5 transposase (Illumina FC-121-1030) for 30 minutes at 37°C, as previously described (Buenrostro et al., 2013), with the modification that fragmented genomic DNA was recovered using Buffer QG (Qiagen 28604). Transposed genomic DNA was amplified by five cycles of quantitative PCR. 10% of the PCR was subjected to an additional 20 cycles of SYBR green-based qPCR while the remainder of the sample was left on ice. Analysis of the qPCR data allowed a rough estimate of the number of additional cycles needed to generate product at 25% saturation. Typically, four to seven additional PCR cycles were added to the initial set of five cycles. Amplified DNA was purified on AMPure XP beads, analyzed on an Agilent Bioanalyzer, and sequenced (paired-end) on an Illumina HiSeq 2500 for 101 cycles. ChIP-seq: Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). ChIP-enriched and input DNA was end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

63830629

Reads aligned (%)

98.1

Duplicates removed (%)

26.7

Number of peaks

288 (qval < 1E-05)

mm9

Number of total reads

63830629

Reads aligned (%)

97.9

Duplicates removed (%)

26.6

Number of peaks

302 (qval < 1E-05)