title: GSM9264382: Pool3: 40, 48, 60, and 72 hpf dissections for ear enrichment; Danio rerio; RNA-Seq
library_name: GSM9264382
library_construction_protocol: To minimize sticking of cells to plastic, 1.5 ml collection tubes were coated with 10% BSA for 10 minutes, rinsed in PBS, and left on ice. Dissections were performed in modified DMEM plus tricaine: 1x DMEM (11039-021 (LifeTech)), 10 mM HEPES, 1x non-essential amino acids (11140076 (LifeTech)), 1mM sodium pyruvate (11360070 (LifeTech), 100 um B-mercaptoethanol (M3148 Sigma), and 0.33 mg/ml Tricaine (E10521 (Sigma)). The morning of an inDrops experiment, a team of 3-4 researchers dissected otic vesicle enriched tissue as in Fig. 1D using two 22-gauge trabecular needles with syringes for handles. For each inDrops run, 40-50 embryos were dissected, and the tissue was collected in a coated collection tube. The DMEM was removed and 50 µl of Trypsin-EDTA was added. Avoiding air bubbles, the tissue was pipetted up and down several times with a 200 µl pipette tip. The tissue was left on ice to dissociate for 3 minutes for 36 hpf tissue, 5 minutes for 48 hpf tissue, or 7 minutes for 60 hpf tissue. We then added 100 µl of FACSMax, pipetted the sample up and down (avoiding air bubbles) and incubated on ice for 2 minutes.  While incubating, we rinsed out a BSA coated tube with PBS. We then added 850 µl of FACSmax to the dissociated cells and pipetted through a 40 µm cell filter into a rinsed BSA coated tube. The samples were spun for 3 minutes at 300g in a cooled swinging bucket centrifuge. Supernatant was removed and cells were resuspended in 1 ml of 0.5% BSA in chilled PBS. We then spun down the cells again for 3 minutes at 300g in a cooled swinging bucket centrifuge. Supernatant was removed and cells were resuspended in 200 µl of 1x prechilled PBS. Cells were counted using a hemocytometer, diluted to 800,000 cells/ml and then used to collect ~15,000 cells by inDrops. Dissection, dissociation, and cell collection was performed twice for each condition. inDrops v3 scRNA-seq
identifiers.namespace: GEO
identifiers.id: GSM9264382_r1