Chn ECJ1 mutant 10x single-cell RNAseq
replicate
1
breed
Canton
age
10-12h
sex
pooled male and female
tissue
whole embryo
genotype
ChnECJ1
growth_protocol
Fly stocks were maintained with standard protocols. Embryos were collected from food plates (corn+yeast extract+sugar+agar) at 25C and used directly for experiments.
treatment
10-12 hour old homozygous embryos of Chn34E and ChnECJ1 mutants were collected along with wild type. Embryos were processed for single cell RNA seq using KARAISKOS et al's protocol (DOI: 10.1126/science.aan3235). In brief, Embryos were dechorionated for 1 min in ~4% NaOCl and extensively washed with deionized water and rinsed with PBT (1xPBS, 0.1% Triton X100). Approximately 200 10-12 hour embryos were collected prior to dissociation. Embryos were washed thoroughly using ice cold cell culture grade PBS and resuspended in 1ml ice cold dissociation buffer (cell culture grade PBS, 0.01% molecular biology grade BSA) and dissociated in a Dounce homogenizer (Wheaton #357544) with gentle, short strokes of the loose pestle on ice until all embryos were disrupted. The suspension was transferred into a 1.5 ml microfuge tube, cells were pelleted for 3' at 1 000g at 4°C. The supernatant was exchanged for 1 ml fresh dissociation buffer. Cells were further dissociated using 20 gentle passes through a 22G x 2” needle mounted on a 5 ml syringe. The cell suspension was then gently passed through a 20 μm cell strainer (Merck, NY2002500) into a fresh 1.5ml microfuge tube, residual cells were washed from strainer using a small amount of dissociation buffer. Cells were counted for viability and processed via 10X with v3 chemistry, then sequenced on the Illumina HiSeq machine.
BioSampleModel
Model organism or animal