|
| Status |
Public on Mar 28, 2019 |
| Title |
GC-JC-7819-G06_S47 |
| Sample type |
SRA |
| |
|
| Source name |
mound stage cell
|
| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX3 with H2B-mCherry inserted in the rps30 locus
replicate number: 3
developmental stage: mound
|
| Growth protocol |
Cells were cultured in petri dishes in HL5 media (Formedium). For development, cells were washed in KK2 (20mM KPO4 pH 6.2) then plated on KK2/1.5% agar in 35mm petri dishes with 1-5 x 106 cells. The dishes were incubated at 22°C for 14h. Aggregates were disaggregated to single cells by taking them up in 1ml KK2/10mM EDTA and repeatedly passing the structures through a 20G needle.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were loaded onto Integrated Fluidic Circuit chips (IFC; Fluidigm). We identified capture of multiple cells and empty wells using brightfield illumination, with validation of single cell capture also carried out using a genetically-encoded red fluorescent nuclear marker. Cell lysis, reverse transcription and cDNA pre-amplification were performed in the C1 Single-Cell Auto Prep IFC using the SMARTer PCR cDNA Synthesis Kit (Clontech) and the Advantage 2 PCR Kit, as specified by the manufacturer (protocol 100-7168 A2). ERCC RNA spike-in control mix (92 transcripts; ThermoFisher) was added to the chambers at a 1:1000 ratio.
cDNA was harvested and the libraries were prepared using the Nextera XT DNA Sample Preparation Kit and the Nextera Index Kit (Illumina), according to the manufacturer's recommendations (protocol 100-7168 A2). Libraries from one chip were pooled, and paired-end 75bp sequencing was performed on 4 lanes of an Illumina NextSeq500.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
processed data file:
Dd_Mound_Rep3.txt
14h3_42
|
| Data processing |
Paired-end reads were mapped to the Dictyostelium genome (version obtained from Gareth Bloomfield, masking the duplication on chromosome 2) using Tophat version 2.0.9 (parameters: -r 300 -p 4 -g 1).
Reads for each gene were counted with htseq-count (parameters: -m union -s no -a 0).
Insert size was calculated with picard 1.119 (CollectInsertSizeMetrics)
Genome_build: dicty_2.7
Supplementary_files_format_and_content: Tab-delimited text files include raw read counts for each replicate.
|
| |
|
| Submission date |
Mar 28, 2019 |
| Last update date |
Mar 29, 2019 |
| Contact name |
Vlatka Antolovic |
| E-mail(s) |
v.antolovic@ucl.ac.uk
|
| Organization name |
University College London
|
| Department |
MRC Laboratory for Molecular Cell Biology
|
| Street address |
Gower Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26360 |
| Series (1) |
| GSE128974 |
Transition state dynamics during a stochastic fate choice |
|
| Relations |
| BioSample |
SAMN11279294 |
| SRA |
SRX5587662 |
