|
| Status |
Public on Nov 03, 2021 |
| Title |
DNeg_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
domeMESO-GFP.nls HmlΔ-DsRed.nls_DNeg
|
| Organism |
Drosophila melanogaster |
| Characteristics |
line: domeMESO-GFP.nls Hml{delta}-DsRed.nls
age: 90-96 hours after egg lay (25 degrees C)
tissue/cell type: lymph gland blood cells
cell population: Double Negative
|
| Treatment protocol |
Experiment was done on untreated wild-type larvae.
|
| Growth protocol |
20 virgin females and 10 males were mated in each vial at 25 degrees C. Eggs were laid in the vials for a 6 hour and then adults were transferred to a new vial. Vials were placed back at 25 degrees for 90-96 hours until larvae reached early thrid instar. Larvae were thoroughly washed in DEPC-treated water prior to dissection.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Lymph glands were dissected out of early 3rd instar larvae, their anterior lobes were removed, and the primary lobes were transferred to a watchglass filled with dissecting buffer on ice. After dissection, the lymph glands were dissociated as previously described with some modifications (Harzer et al. 2013; Khan et al. 2016). After being washed with MDS buffer twice, these tissues were transferred to 1.5 mL DNA LoBind tubes (Eppendorf) and incubated with 200 µL of dissociation solution containing 1 mg/mL of papain (Sigma, P4762) and 1 mg/mL of collagenase (Sigma, C2674) in Schneider’s medium. They were dissociated for 15 minutes in a shaking incubator at 25°C, 300 rpm. Next, 500 µL of cold Schneider’s medium was added and the suspension was gently pipetted up and down using a low-binding 1000 µL tip (Olympus Plastics) 20 times for mechanical dissociation. After centrifugation at 3000 rpm for 5 minutes, cells were resuspended and washed with 500 µL of 1X PBS (Corning, MT21040CV) containing 0.04% of UltraPure BSA (Invitrogen, AM2616) and then passed through a 35 micrometer cell strainer (Falcon 352235). , Dissociated, live lymph gland cells were sorted using the BD FACSARIA-H. Gates and compensation were based on single color controls. Cells were sorted into 300 µL of DNA/RNA Shield (Zymo) in DNA LoBind tubes and frozen at -80°C prior to RNA extraction. Total RNA was extracted from each sample using the Quick-RNA Microprep Kit (Zymo). RNA quality control was performed using the Agilent 4150 TapeStation system.
3' mRNA capture: cDNA libraries were prepared using KAPA Stranded mRNA-seq kit (KAPA Biosystems)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
Double Negative biological replicate 1
dnlsDNeg1_S10
|
| Data processing |
Illumina software for basecalling
Sequencing data was analyzed using Partek Flow, a web-based software platform. Sequences were aligned to the Drosophila melanogaster reference genome r6.22 (Flybase) using STAR aligner with default parameters. Read counts were normalized by counts per million (CPM). Differential genes expression analysis was performed using ANOVA with a fold change cutoff of 2 and FDR<0.05 (see Supplementary file 3 Tables 1 and 2).
Genome_build: r6.22
|
| |
|
| Submission date |
Mar 12, 2021 |
| Last update date |
Nov 03, 2021 |
| Contact name |
Juliet Girard |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California Los Angeles
|
| Street address |
610 Charles E Young Dr E
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL23323 |
| Series (2) |
| GSE168821 |
Bulk RNA sequencing on sorted blood cells from Drosophila lymph glands [MZ IZ bulk RNA-seq] |
| GSE168823 |
Paths and Pathways that Generate Cell-Type Heterogeneity and Developmental Progression in Hematopoiesis |
|
| Relations |
| BioSample |
SAMN18287685 |
| SRA |
SRX10332290 |
