title: GSM9609625: CC fate marker in non-reprogramming and reprogramming CCs, rna_aer030, L3, Repro_CC; Caenorhabditis elegans; RNA-Seq
library_name: GSM9609625
library_construction_protocol: The protocol by Kaletsky et al., 2016 (Kaletsky, et al. 2016) was used with modifications to dissociate coelomocyte cells from L3 worms. Pelleted animals were washed with 1-2x volume of lysis buffer, pulse spun and the supernatant was removed. Lysis buffer was then added up to ~4-10x volume of the animal pellet, and the lysis reaction was run for 6.5 minutes at RT whilst rotating at 20 rpm. 2x volume of ice-cold M9 was added, and the sample was spun at 1000 g for 30 seconds at 4 °C. The supernatant was removed, and the resulting pellet was washed a further 6 times. M9 was then added up to 200 uL and mixed in a 1:1 ratio with pronase (20 mg/mL, warmed at 37 °C) at RT. 1x volume of chitinase (1 U/mL) was added, and the L3-chitinase mix was spun at 1300 g for 1 minute. The supernatant was then removed, and 4x volume chitinase (1 U/mL in egg buffer) was added and incubated at RT, rotating at 20 rpm for 1 hour. 2.5 volumes of PBS+2% FBS were then added to stop the reaction, and chitinase-treated worms were spun at 900 g for 3 minutes at 4 C. The supernatant was removed, and PBS+2% FBS was added up to 400 uL. For mechanical dissociation, 10 minutes of pipetting per sample, with a 1-minute break between each minute of pipetting, was applied. The pipette was pressed against the bottom of the tube whilst sucking up liquid and samples were aspirated into the remaining sample. A 10-um filter was wetted with PBS + 2% FBS and used to filter the dissociated cells. The filtered cells were pelleted at 1000 g for 5 min at 4 °C, and the supernatant was removed. Dissociation by pipetting and preparation for FACs sorting was then performed as described for the dissociation of embryos. Dissociated cells were resuspended in 1x PBS and filtered through a Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap (35 mm nylon net). Cells were sorted out using either a BD FACS ARIA III or BD FACSARIA FUSION FACs machine. Sorted cells were collected in a 1.5 mL Eppendorf tube containing 50 mL PBS and the RNase inhibitor Ribolock at a final concentration of 1 U/mL.Samples were diluted 1 in 10 with Trizol (15596026 LIFE technologies) and glycoblue was added at 1 to 1000 to ensure that RNA pellets were visible following isolation. Samples were then vortexed for 30 seconds, incubated at RT for 5 mins and flash frozen in liquid nitrogen. 0.2x volume of chloroform was added to the sample, followed by 30 seconds of vortexing and 5 minutes of incubation at RT. Samples were then centrifuged at 12,000 g for 15 minutes at 4 °C. The clear aqueous layer was isolated and an equal volume of isopropanol was added. Samples were then inverted ten times and kept at -20 °C overnight. Samples were then centrifuged at 12,000 g for 30 minutes at 4 °C and the supernatant was discarded. The RNA + glycoblue pellet was then washed with 1 mL 75% ethanol, shaken briefly, and centrifuged at 7,500 g for 5 minutes at 4 °C. The supernatant was then discarded, and pellets were air-dried for 10 minutes at RT. RNA was resuspended in 20-25 mL RNase-free water. Poly(A) RNA was selected for using the NEXTflex™ Poly(A) Beads Kit (BIOO SCIENTIFIC) according to the manufacturer's instructions. RNA-seq libraries were prepared using the NEXTflex™ Rapid Directional qRNA-Seq™ Kit (BIOO SCIENTIFIC) according to the manufacturer's instructions.