|
| Status |
Public on Sep 30, 2021 |
| Title |
CLTP3 |
| Sample type |
SRA |
| |
|
| Source name |
rat hippocampal culture 3 with induced cLTP
|
| Organism |
Rattus norvegicus |
| Characteristics |
agent: induced cLTP
tissue: hippocampal
|
| Treatment protocol |
Chemically-induced long-term potentiation of synaptic transmission (cLTP) was induced according to previously published protocol (Szepesi et al., 2013) by adding 50 μM forskolin (Sigma Aldrich), 50 nM rolipram (Sigma Aldrich), and 200 μM picrotoxin (Sigma Aldrich) to the medium on DIV13-15. As the chemicals were dissolved in DMSO, the same amount of the solvent was added to the medium of the control.
|
| Growth protocol |
Primary hippocampal neurons from Wistar rat pups at P0 were cultured in 6-well plates (VWR) or in 24-well plates (VWR) on 12 mm glass coverslips (VWR) coated with poly-D-lysine (Sigma Aldrich) and laminin (Roche)
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using RNAeasy Mini Kit (Qiagen), according to the company's recommendations (a detailed protocol can be found on the producers website). Briefly, cells from the 6-well plates were disrupted in Buffer RLT and transferred to the RNAse-free tube. RNA was then purified on the spin column, including DNase I step, and eluted with 30 μL of nuclease-free water. Purity and concentration of RNA were measured using NanoDrop One (Thermo Scientific).
In total, 6 strand-specific RNA libraries for high-throughput sequencing were prepared (three biological replicates for each treatment) using the KAPA Stranded mRNA Sample Preparation Kit according to the manufacturer’s protocol. Briefly, the poly-A containing mRNA molecules were purified from 500ng of total RNA (extracted FACS sorted cells from the brain samples of sham operated and MCAo animals) using poly-T oligo-attached magnetic beads (Kapa Biosystems, MA, USA ) The mRNA was then fragmented and the first-strand cDNA was synthesized using reverse transcriptase and random hexamers. Second cDNA synthesis was performed by removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP, to generate double-stranded (ds) cDNA. dsDNA was then subjected to the addition of “A” bases to the 3′ ends and ligation of adapters from NEB followed by uracil digestion in a loop structure of adapter by USER enzyme from NEB (Ipswich, MA, USA). Amplification of fragments with adapters ligated on both ends was performed by PCR using primers containing Truseq barcodes from NEB (Ipswich MA, USA).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
Basecalling with HCS 2.2.68
Bcl2Fastq processing of bcl files to fastq
Fastq trimming for adapter contamination and low quality reads and aligned with STAR to rn6.fa reference
DESeq2 normalization and statistical analysis
Genome_build: rn6
Supplementary_files_format_and_content: DESeq2 normalized data table, normalized for library size and gene length
|
| |
|
| Submission date |
Feb 11, 2021 |
| Last update date |
Sep 30, 2021 |
| Contact name |
Bartosz Wojtas |
| E-mail(s) |
bartoszwojtas83@gmail.com
|
| Phone |
692156006
|
| Organization name |
Nencki Institute of Experimental Biology
|
| Street address |
Pasteura 3
|
| City |
Warsaw |
| ZIP/Postal code |
02-793 |
| Country |
Poland |
| |
|
| Platform ID |
GPL18404 |
| Series (1) |
| GSE166599 |
The changes of the nuclear landscape upon stimulation of neuronal cells are dependent on the histone deacetylase HDAC1 |
|
| Relations |
| BioSample |
SAMN17864918 |
| SRA |
SRX10075005 |
