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| Status |
Public on May 31, 2022 |
| Title |
Cux2_E18_5_R1 |
| Sample type |
SRA |
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| Source name |
somatosensory cortex and motor cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: somatosensory cortex and motor cortex
genotype: Cux2-CreERT2:Ai14
treatment: 4-HydroxyTamoxifen injection
age: E18.5
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| Treatment protocol |
4-Hydroxytamoxifen (4-OHT; Sigma) dissolved in corn oil was administered to pregnant mice at 1 mg 4-OHT/10g body weight.
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 mice were conducted under protocols approved by the Harvard University 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 |
total RNA |
| 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.
Pools of 5,000-10,000 cortical pyramidal neurons were sorted directly into TRIzol-LS buffer (Invitrogen) and RNA was extracted according to the manufacturer’s protocol. 10 ng total RNA was used for library preparation using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) and the Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw reads were trimmed, removing 8bp from the 5’ and 25 bp from the 3’ end.
Reads were aligned to the mm9 reference genome using Tophat2
Subsequently, differential gene expression analysis and FPKM quantification was performed for all pairwise comparisons
Assembly: mm9
Supplementary files format and content: tab-delimited text file includes FPKM values for each Sample
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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 |
GPL17021 |
| Series (2) |
| GSE204757 |
Temporally-Divergent Regulatory Mechanisms Govern Neuronal Diversification and Maturation in the Neocortex [RNA-seq] |
| GSE204851 |
Temporally-Divergent Regulatory Mechanisms Govern Neuronal Diversification and Maturation in the Neocortex |
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| Relations |
| BioSample |
SAMN28647855 |
| SRA |
SRX15446234 |
| 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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