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| Status |
Public on May 31, 2022 |
| Title |
Day0.rep3.calJac3 |
| Sample type |
SRA |
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| Source name |
somatosensory cortex
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| Organism |
Callithrix jacchus |
| Characteristics |
tissue: somatosensory cortex
genotype: Wild type
age: Day0
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| Treatment protocol |
non
Cortex was dissected in cold dissociation medium (20 mm glucose, 0.8 mm kynurenic acid, 0.05 mmAPV, 50 U/ml penicillin–0.05 mg/ml streptomycin, 0.09mNa2SO4, 0.03mK2SO4, and 0.014m MgCl2). The cortex was enzymatically digested in dissociation medium containing 0.16 gm/l l-cysteine and 12 U/ml papain at 37°C for 30 min. Papain digestion was then blocked with dissociation medium containing 10 mg/ml ovomucoid (Sigma, St. Louis, MO) and 10 mg/ml bovine serum albumin (BSA) at room temperature. Neurons were mechanically dissociated to create a single cell suspension by gentle trituration in iced OptiMem (Life Technologies, Gaithersburg, MD) containing 20 mm glucose and both 0.4 mmkynurenic acid and 0.025 mm APV to protect against glutamate-induced neurotoxicity. Td-tomato labeled projection neurrons were purified from the cortical cell suspension by fluorescence-activated cell sorting (FACS) using a FACSVantage flow cytometer.
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| Growth protocol |
Experiments using animal were conducted under protocols approved by the Harvard University and MIT Institutional Animal Care and Use Committee and followed the guidelines set forth in the National Institute of Health Guide for the Care and Use of Laboratory Animals.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To prepare nuclei, around 50,000 cells at 500g for 5 min, which was followed by a wash using 50 μL of cold 1× PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge.
For mouse: Briefly, cells were fixed with 0.1% formaldehyde and incubated at room temperature for 5 min. The fixation was stopped by adding glycine to the final concentration of 125 mM. The sample was incubated at room temperature for 5 min and washed in PBS. The cell concentration was counted, and approximately 1600-2000 cells per well were distributed into each well of a 96-well plate. Cells were transposed with 96 uniquely barcoded Tn5 at 37°C for 30 min with shaking at 300 rpm. The reaction was stopped by adding 0.5 M EDTA and incubated at 37°C for 15 min. All the cells were then pooled and MgCl2 was added to the pooled sample to quench EDTA. The sample was re-distributed onto another 96-well plate with 20 cells in each well by FACS sorting. Reverse crosslinking buffer and barcode PCR primers were added to each sample. The plate was incubated at 55°C for 16 hours for reverse crosslinking. Tween 20 was then added to quench SDS before PCR amplification. The PCR reaction was carried out at the following conditions: 72°C for 5 min (extension), 98°C for 5 min, and then thermocycling at 98°C for 10 s, 70°C for 30 s and 72°C for 1 min for 12-15 cycles. Libraries were pooled and purified using Qiagen MinElute PCR purification column. The libraries were quantified using KAPA library quantification kit. Libraries were sequenced on the Next-seq platform (Illumina) using a 150-cycle kit (Read 1: 47 cycles, Index 1: 36 cycles, Index 2: 36 cycles, Read 2: 47 cycles). For marmoset: Somatosensory and motor cortex from wild-type animals was dissected and dissociated with the Worthington Papain Dissociation System (Worthington Biochemical Corporation), and live cells were isolated by FACS sorting as DAPI-negative, Vybrant DyeCycle Ruby (Thermo Fisher)-positive events. Libraries were prepared using the 10x Genomics Chromium Single Cell ATAC kit (10x Genomics).
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Mouse and marmoset libraries were first downsampled to similar overall complexity in terms of reads/cell across all conditions
Cleaned data was processed with the scasat83 pipeline using macs2 as peak caller
Only cells with at least 1,000 reads and 300 peaks from the master list overlapping with at least one read, but not more than 15,000 reads and 8,000 peaks were retained for analysis
matrix was processed using R implementing a custom processing pipeline based on the strategy outlined in Cusanovich et al. 2018 and refined by Andrew Hill, following the log-LSI workflow
Assembly: mm10, calJac3
Supplementary files format and content: gene-cell matrix with raw counts (RDS format) and tab-delimited text file with cell metadata
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| Submission date |
May 24, 2022 |
| Last update date |
Jun 07, 2022 |
| Contact name |
Paola Arlotta |
| E-mail(s) |
paola_arlotta@harvard.edu
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| Organization name |
Harvard University
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| Department |
Stem Cell and Regenerative Biology
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| Lab |
Arlotta
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| Street address |
7 Divinity Ave
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL21580 |
| Series (2) |
| GSE204761 |
Temporally-Divergent Regulatory Mechanisms Govern Neuronal Diversification and Maturation in the Neocortex [scATAC-seq] |
| GSE204851 |
Temporally-Divergent Regulatory Mechanisms Govern Neuronal Diversification and Maturation in the Neocortex |
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| Relations |
| BioSample |
SAMN28647946 |
| SRA |
SRX15446588 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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