title: GSM4832539: NM-AS_ST5_E-2; Capitella teleta; RNA-Seq
library_construction_protocol: For single-cell dissociation, 300 larvae from a single brood (i.e. from one male and female) for each stage were then collected into 1.5 mL centrifuge tubes and equilibrated in Ca2+/Mg2+-free ASW (CMFSW). Most of the CMFSW was removed from the tubes, and larvae were mechanically homogenized using separate, clean and sterile pestles as well as a hand-held homogenizer (Cole Parmer, LabGEN 7B) for 5–10 seconds. Homogenized larvae from each stage were then incubated in 1% Trypsin (SigmaAldrich, Cat# T4799-5G) in CMFSW for 30 minutes at room temperature with constant rocking. During incubation, dissociated tissues were periodically triturated using both wide-mouthed and narrow-mouthed Pasteur pipettes. After 30 minutes of incubation, the tissue lysate was passed through a 40 µm nylon cell-strainer (Fisherbrand, Cat# 22-363-547) to get rid of undissociated cell-clumps. The resultant cell-suspension was then centrifuged at 1100 x g for 7 minutes with slow-braking and washed twice in cell media that was developed originally for marine hemichordate cell-cultures (3.3X Dulbecco's PBS and 20 mM HEPES, pH = 7.4). Dissociated cells were then resuspended in the cell media and checked under an inverted phase-contrast microscope to ensure a single-cell suspension was obtained. RNA libraries were prepared for sequencing using standard Illumina protocols for NextSeq-500 paired-end RNAseq