Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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

Adult human retina

donor apparent sex

Male

donor age

60

donor apparent race

caucasian

postmortem interval

7:38

chip_seq antibody

Abcam cat# ab7766 lot GR160184_1; RRID: AB_2560996

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

METHOD DETAILS: DNA accessibility: ATAC_Seq: Flash_frozen tissue was thawed by addition of 700ul of lysis buffer (10 mM Tris_HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP_40), homogenized by trituration 10x with a p1000 pipet set to 500ul, dounced in an ice cold RNase_free 2mL glass dounce 10x with a loose pestle and 10x with tight pestle, and transferred back to original 1.5mL LoBind tube (Eppendorf). Dounce was washed with an additional 700ul ice cold lysis buffer and buffer was transferred to tube with sample for a final volume of 1.4mL. Sample was then centrifuged in a microcentrifuge at 4C for 10min at 500 rcf. The pellet was re_eluted in 1.4mL ice cold lysis buffer, transferred to a pre_chilled dounce, homogenized again 12x with tight pestle, and transferred back to 1.5mL LoBind tube. Nuclei were counted on a hemocytometer and the volume of lysis buffer/nuclei suspension needed for 20,000 nuclei was aliquoted into a separate 1.5mL LoBind tube. Samples were centrifuged along with tube containing remaining nuclei (for nuclear localized RNA) at 4C for 5min at 500g (RCF). The supernatant was removed from samples and remaining nuclei. Subsequent ATAC libraries were generated with 20,000 nuclei per sample using the Nextera DNA library prep kit (FC_121_1030; Illumina, San Diego, CA USA) according to the protocol described in (Buenrostro et al., 2013; Buenrostro et al., 2015). DNase I_Seq: Sequence files from the ENCODE Consortium (Consortium, 2012) for human retinal pigment epithelium (ENCBS420ENC, ENCBS893GDP), embryonic human retina (ENCSR820ICX, ENCSR474GZQ, ENCSR621ENC), and 8 week mouse retina (ENCSR000CNW) described in (Wilken et al., 2015) were analyzed in parallel with ATAC_Seq data. Chromatin immunoprecipitation: Crosslinking: Flash frozen human or mouse tissue was homogenized in crosslinking buffer (10 mM HEPES_NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) containing 1% formaldehyde (added immediately before crosslinking) by trituration 10x with a p1000 pipet set to 500ul, dounced in an ice cold RNase_free 2mL glass dounce 10x with a loose pestle and 10x with tight pestle, transferred back to original 1.5mL LoBind tube (Eppendorf), and incubated while rotating gently at room temperature for 10 min. Crosslinks were quenched by addition of glycine to a final concentration of 0.125 M and incubated while rotating gently at room temperature for 5 min. Cells were pelleted by centrifugation 5 min at 1,350 g at 4C. Cell pellets were resuspended once with cold PBS and re_spun for 5 min at 1,350 g at 4C. Nuclei prep: Crosslinked cell pellets were resuspended in cold 5mL L1 buffer (50 mM HEPES_NaOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.25% Triton X_100, 0.5% NP_40, 10% glycerol, protease inhibitors; 10 mM sodium butyrate added for H3K27ac ChIPs) by pipetting and rotated vertically at 4C for 10 min. Pellets were spun for 5 min at 1,350 g at 4C, and the supernatants were aspirated. Pellets were resuspended in cold 5mL L2 by pipetting (10mM Tris_HCl pH 8.0, 200mM NaCl, protease inhibitors; 10 mM sodium butyrate added for H3K27ac ChIPs) and rotated vertically at 4C for 10 min. Pellets were spun for 5 min at 1,350 g at 4C, and the supernatants were aspirated. Pellets were resuspended in cold 1.5 mL LB3 (10mM Tris_HCl pH 8.0, 100mM NaCl, 1mMEDTA, 0.5mMEGTA, 0.1% sodium deoxycholate, 0.5% N_lauroylsarcosine, protease inhibitors; 10mM sodium butyrate added for H3K27ac ChIPs) by pipetting and transferred to polystyrene tubes (Thermo Fisher 352099) for sonication. Sonication: Nuclei pellets were sonicated (Bioruptor Plus, Diagenode) in polystyrene tubes on high power with 36 cycles of 30 sec ''on'', 45 sec ''off''. After sonication, Triton X_100 was added to 1% final concentration and sonicated chromatin was centrifuged at 16,000 g for 5 min at 4C. The supernatant was used for preclearing and ChIP. All subsequent steps were performed using DNA LoBind tubes (Eppendorf). Preclearing and antibody_bead coupling: Protein A Dynabeads (Life Technologies) were washed twice with blocking buffer (0.1% BSA in LB3 + 1% Triton X_100) and aliquoted for preclearing and antibody_bead coupling. Antibodies were coupled to beads in 1.8 mL of blocking buffer by vertical rotation at 4C for 4 hr. In parallel, each sample of sonicated chromatin was incubated with an equivalent volume of washed bead slurry for preclearing. Each ChIP was performed in 1.8 mL LB3 + 1% Triton X_100 and rotated vertically at 4C for 16 hr. For input samples, 1% of sample was saved for de_crosslinking and DNA purification. Washes and elution: For each wash, beads were rotated vertically in wash buffer at 4C for 5 min. Beads were washed twice with low salt wash buffer (0.1% SDS, 1% Triton X_100, 2 mM EDTA, 20 mM Tris_HCl pH 8.0, 150 mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X_100, 2 mM EDTA, 20 mM Tris_HCl pH 8.0, 500 mM NaCl), twice with lithium chloride wash buffer (0.25 M LiCl, 1% NP_40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris_HCl pH 8.0), and once with TE (50 mM Tris, 10 mM EDTA). Beads were then incubated at 65C in 200 mL of TE + 1% SDS per sample for 30 min with vortexing every 10 min. Eluted protein_DNA complexes were separated from the beads and incubated at 65C for 16 hr to reverse crosslinks. Purification of immunoprecipitated DNA: Elutions were incubated with 10mg RNase A for 1 hr at 37C, followed by 140 mg proteinase K for 2_3 hr at 55C with shaking. DNA was extracted with 1 volume of 25:24:1 phenol_chloroform_isoamyl alcohol and purified with a Qiagen PCR purification kit. ChIP DNA concentrations were determined by Qubit dsDNA HS Assay Kit (Thermo Fisher). Gene expression: Total RNA Nuc_seq: To determine RNA expression in adult human retina, macula and RPE nuclei, RNA was extracted from excess nuclei (~80M) from the ATAC nuclear preparation protocol using Trizol (ThermoFisher; 15596026). Prior to RNA extraction, an equal amount of ERCC spike_in control RNA (Mix 2) was added to each sample, following the manufacturer's specifications. RNA was extracted using the Qiagen RNeasy Mini Kit with on_column DNase I digestion (Qiagen 79254). Single Nucleus RNA_seq: Generation of single_nucleus suspensions were prepared by isolating nuclei from flash frozen human retina as described above and purified using an iodixanol gradient (Optiprep; Sigma_Aldrich, St. Louis, MO, USA) with RNase_inhibitor. After gradient centrifugation, the nuclei were washed in dissociation solution (Hrvatin et al., 2018; Klein et al., 2015) containing 0.04% BSA and were resuspended in dissociation solution containing 0.04% BSA and 15% Optiprep for single_nucleus RNA sequencing using a modified inDrops protocol (Klein et al., 2015). Library preparation and sequencing: ATAC_seq: ATAC_Seq libraries were prepared as described above and sequenced on the Illumina NextSeq 500 platform with 75 bp single_end reads, or 2 x 150 bp paired_end reads. ChIP_seq: 2_40 ng of each ChIP sample was used to prepare libraries with the NuGEN Ovation Ultralow Library System v2 kit per manufacturer's protocol. Libraries were sequenced on the Illumina NextSeq 500 platform with 75 bp single_end reads, or 2 x 150 bp paired_end reads.Total RNA Nuc_seq: RNA_seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (E7420L, NEB) and either NEB rRNA depletion kit (E6310, NEB) or Qiagen GeneRead rRNA Depletion Nano kit (180211, Qiagen). Spike_in RNA (Fisher 4456740) were used for sample_to_sample normalization. Samples were then sequenced on the Illumina NextSeq 500 platform with 2x 150 bp paired_end reads. Single_nucleus RNA_seq: Single_nucleus RNA sequencing (inDrops): One or two libraries of approximately 3,000 cells were collected from each donor retina. inDrops was performed as previously described (Klein et al., 2015; Zilionis et al., 2017), generating indexed libraries that were then pooled and sequenced on the NextSeq 500 (Illumina) platform.

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

45696685

Reads aligned (%)

93.9

Duplicates removed (%)

12.5

Number of peaks

50937 (qval < 1E-05)

hg19

Number of total reads

45696685

Reads aligned (%)

93.1

Duplicates removed (%)

12.6

Number of peaks

50211 (qval < 1E-05)