|
| Status |
Public on Dec 03, 2019 |
| Title |
E16_green_Replicate 6 [94756] |
| Sample type |
SRA |
| |
|
| Source name |
E16_green
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD1
age: E16
color: green
tissue: cortex
cre: Emx1
madm: Chr. 7
replicate: Replicate 6
|
| Treatment protocol |
Pregnant females were sacrificed and E13/E16 embryos were collected. Neocortex area was dissected. Single individuals were used as replicates. Single cell suspensions were prepared by using Papain containing L-cysteine and EDTA (vial 2, Worthington), DNase I (vial 3, Worthington), Ovomucoid protease inhibitor (vial 4, Worthington), EBSS (Thermo Fisher Scientific), DMEM/F12 (Thermo Fisher Scientific), FBS and HS. All vials from Worthington kit were reconstituted according to the manufacturer’s instructions using EBSS. The dissected brain area was directly placed into Papain-DNase solution (20units/ml papain and 1000 units DNase). Enzymatic digestion was performed for 30min at 37°C in a shaking water bath. Next, solution 2 (EBSS containing 0.67mg Ovomucoid protease inhibitor and 166.7 U/ml DNase I) was added, the whole suspension was thoroughly mixed and centrifuged for 5min at 1000rpm at RT. Supernatant was removed and cell pellet was resuspended in solution 2. Trituration with p1000 pipette was performed to mechanically dissolve any remaining tissue parts. DMEM/F12 was added to the cell suspension as a washing solution, followed by a centrifugation step of 5min with 1500rpm at RT. Cells were resuspended in media (DMEM/F12 containing 10% FBS and 10% HS) and kept on ice until sorted. Right before sorting, cell suspension was filtered using a 40µm cell strainer. FACS was performed on a BD FACS Aria III using 100 nozzle and keeping sample and collection devices (0.8ml PCR tubes) at 4°C. Duplet exclusion was performed to ensure sorting of true single cells. Cells were sorted into 4µl lysis buffer (0.2% Triton X-100, 2U/µl RNase Inhibitor [Clonetech]). Immediately after sorting was completed samples were transferred into a 96 well plate (Bio-Rad) that was kept on dry ice. GFP+, tdT+ and GFP+/tdT+ cells (200-400 cells) were collected.
|
| Growth protocol |
Mouse protocols were reviewed by institutional preclinical core facility (PCF) at IST Austria and all breeding and experimentation was performed under a license approved by the Austrian Federal Ministry of Science and Research in accordance with the Austrian and EU animal laws. Mice were maintained and housed in animal facilities with a 12-hour day/night cycle and adequate food/water conditions according to IST Austria institutional regulations. Mouse lines with chromosome 7 MADM cassettes: MADM-7-GT JAX stock # 021457, MADM-7-TG JAX stock # 021458, Emx1-Cre: JAX stock # 005628. All MADM-based analyses were carried out in a mixed C57BL/6J, CD1 genetic background.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
sorted cells were directly processed without RNA extraction
Smart-Seq2 (VBCF GmbH Vienna)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
94756_ACCTTATGAATGTTAGAAGG.CDNR1ANXX
|
| Data processing |
All libraries were sequenced 3x (technical replicates) to increase read depth. Raw reads were delivered as unaligned BAM files and technical replicates were combined using samtools (v1.8) and converted to fastq format using bamToFastq (bedtools suite v2.26.0) for alignment. Reads within transcripts were counted using STAR_2.5.0c with an index prepared from GRCm38 and Gencode M16 gene annotation. Note that for optimal representation of the long non-coding RNA Meg3 the spliced ENSMUST00000143836.7 transcript was modified to represent a single exon transcript. Parameters used for STAR alignment: --outFilterMultimapNmax 1 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --quantMode GeneCounts
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Read counts in transcripts as reported by STAR as tab delimited text
|
| |
|
| Submission date |
Oct 01, 2019 |
| Last update date |
Dec 05, 2019 |
| Contact name |
Florian M Pauler |
| E-mail(s) |
florian.pauler@ista.ac.at
|
| Organization name |
Institute of Science and Technology Austria (IST Austria)
|
| Lab |
Hippenmeyer
|
| Street address |
Am Campus 1
|
| City |
Klosterneuburg |
| ZIP/Postal code |
3400 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE138227 |
Imprinted Cdkn1c Genomic Locus Cell-Autonomously Promotes Cell Survival in Cerebral Cortex Development |
| GSE138230 |
Imprinted Cdkn1c Genomic Locus Cell-Autonomously Promotes Cell Survival in Cerebral Cortex Development |
|
| Relations |
| BioSample |
SAMN12879283 |
| SRA |
SRX6929298 |
