 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 21, 2023 |
| Title |
NL_CN_1_IgG_CnR |
| Sample type |
SRA |
| |
|
| Source name |
Brain
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Brain
cell type: Patient-derived post-mortem brain tissue from caudate nucleus
genotype: Clinically healthy
antibody: Sigma I8144
|
| Growth protocol |
We sectioned tissue from the caudate nucleus into aliquots of ~100 mg. We performed sectioning on dry ice with sterile forceps and a sterile, single-use razor using a petri dish as a platform after all equipment had been pre-chilled on dry ice. Prior to douncing and homogenization, we pre-chilled all buffers, reagents, and equipment on wet ice. We performed the entire procedure on wet ice. We placed tissue in 10 mL of ice-cold Homogenization Buffer (0.32 M sucrose (Sigma-Aldrich, S0389-500G), 5 mM CaCl2 (Thermo Fisher, J63122-AD), 10 mM Tris-HCl pH 8.0 (Invitrogen, 15568025), 3 mM MgAc2 (Sigma-Aldrich, 63052-100ML, 0.1% Triton X-100 (Sigma-Aldrich, T8787-100ML), 0.1 mM EDTA (Invitrogen, 15575020), 1X Protease Inhibitor Cocktail (Roche, 11873580001)) and dounced the tissue with 20 strokes of the loose pestle and 7 strokes of the tight pestle using a 15 mL Dounce Tissue Grinder (Wheaton, 357544). We performed douncing very slowly and gently to avoid unnecessary mechanical stress. We laid 10 mL of homogenized tissue over 14 mL of ice-cold Sucrose Cushion (1.8 M sucrose, 10 mM Tris-HCl pH 8.0, 3 mM MgAc2, 1X Protease Inhibitor Cocktail). We laid an additional 12 mL of ice-cold Homogenization Buffer on top of the homogenized tissue and centrifuged for 2 hours at 4°C at 25,700 RPM (~81,150xg) in a SW Ti 32 swinging bucket rotor. We removed the supernatant and added FANS Buffer (1X PBS (Corning, 21-040-CV), 1% Bovine Serum Albumin (Sigma-Aldrich, A7906-50G), 1X Protease Inhibitor Cocktail), to the pellet. We incubated the pellet on ice for 20 mins before resuspending. We counted the nuclei and centrifuged the solution for 6 mins at 4°C at 600xg. We resuspended nuclei in FANS buffer at a concentration of 3 million nuclei per mL. We blocked nuclei in FANS buffer for 15 mins at 4°C while rotating. We stained nuclei with anti-NeuN (1:1000, Sigma-Aldrich, MAB377X) for 90 mins and added DAPI (1:2000, Sigma-Aldrich, MBD0015-1ML) with 5 mins left on the staining timer. Staining was performed with end-over-end rotation at 4°C. Next, we centrifuged the nuclei for 6 mins at 4°C at 600xg. We resuspended nuclei in FANS buffer at a concentration of 6 million nuclei per mL, filtered the solution using a 5 mL FACS sorting tube (Corning, 352235), and sorted using the MoFlo Astrios (Beckman Coulter). All tissue samples were from male patients.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN was completed as previously described (Epicypher). In brief, iPS cells were harvested and then washed in wash buffer (20 mM Hepes KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermadine, 1 Roche Complete Protease Inhibitor EDTA-free mini tablet per 10 mL) and bound to Concanavalin A beads (BioMagPlus) that had been activated and washed with binding buffer (20 mM Hepes KOH pH 8.0, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). The cells were then incubated with the Concanavalin A magnetic beads, primary antibody (either IgG (Sigma I8140) or H3K9me3 (Abcam ab8898), and antibody buffer (digi-wash buffer – 0.1% digitonin in wash buffer – with 2 mM EDTA) overnight at 4 C while rotating. Cells were washed with digi-wash buffer and then incubated in a solution containing protein A-MNase and digi-wash buffer for 10 minutes at room temperature. After incubation, the samples were washed in digi-wash buffer and 100 uL digi-wash buffer was added to the samples which were then placed on ice to chill for five minutes. After chilling, 2 uL of 100 mM CaC was added to activate protein A-MNase chromatin digestion. After 2 hours at 4 C with rotation, 100 uL of 2X stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 50 ug/mL RNase A, 50 ug/mL Glycogen) was added to halt the reaction which was then incubated at 37 C for 30 minutes to release chromatin fragments. Supernatant was collected and DNA was extracted using phenol-chloroform and ethanol precipitation. The resulting DNA was quantified on a Qubit Fluorometer. For nuclei isolated from brain tissue, slight modifications were made. We performed CUT&RUN on sorted nuclei as described below with minor modifications. We sorted nuclei into CUT&RUN Wash Buffer and immediately bound nuclei to Concanavalin A beads after returning from sorting. Additionally, we substituted 0.1%, 0.1%, and 0.05% digitonin in the Antibody Buffer, Digi-Wash Buffer, and 2X Stop Buffer with 0.1%, 0.1%, and 0.04% Triton X-100. All other steps were the same.
CUT&RUN libraries were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit (NEB #7103) according to manufacturer's protocol. Size selection of adaptor-ligated libraries was performed using AgenCourt Ampure XP beads (Beckman Coulter, A63881) according to the manufacturer’s protocol. Size selection targeted <1 kb fragment size and libraries were amplified using 11 PCR cycles. Input amounts for library preparation using the NEBNext Ultra II DNA Library Prep Kit were 1 ng of purified libraries. Library quality and quantities were assessed using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
We analyzed CUT&RUN sequencing data using Bowtie2 (version 2.2.5) with parameters “--local --very-sensitive-local --no-mixed --no-discordant --phred33 -I 10 -X 700”. We removed duplicates and unmapped reads and then converted the file to bam format using Samtools (version 1.11) fixmate, sort, markdup “-r”, and view “-F 4” commands. We downsampled mapped reads for IgG and H3K9me3 samples to the lowest number of mapped reads for each comparison group using Samtools view with parameters “-hbs” and a seed of 42. Indices were created for each file using Samtools “index”. We then input normalized bam files using BamCompare from deeptools (version 3.3.0) using the “–extendReads –binSize 10 –smoothLength 30 –operation subtract” parameters. For CUT&RUN data from brain tissue, we performed data processing as earlier described with minor modifications. After mapping, we kept duplicates instead of removing them. Unlike with ChIP-seq, this is an acceptable method of data processing. In CUT&RUN, targeted DNA fragmentation is performed using a pA/G-MNase fusion protein tether to an antibody which is bound to the target. As a result, duplicates are expected based on MNase cutting DNA in a non-random pattern. This is unacceptable in ChIP-seq as DNA is randomly sheared and therefore, duplicates are expected to be primarily from PCR over-cycling. Lastly, we converted downsampled bam files to bigwigs with log2 input normalization using BamCoverage from Deeptools (v3.3.0) with parameters “--extendReads –binSize 10 –smoothLength 30 --operation log2”. All other steps were the same.
Assembly: hg38
Supplementary files format and content: The CUT&RUN processed data files are in the ENCODE bigwig and BED format.
Library strategy: CUT&RUN
|
| |
|
| Submission date |
Jul 17, 2023 |
| Last update date |
Dec 21, 2023 |
| Contact name |
Jennifer E Phillips-Cremins |
| E-mail(s) |
jcremins@seas.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Bioengineering
|
| Street address |
415 Curie Blvd
|
| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE218677 |
Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome [CUT&RUN] |
| GSE218680 |
Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome |
|
| Relations |
| BioSample |
SAMN36504420 |
| SRA |
SRX21050390 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
