GSM6339356: Batch4_unsorted_ethanol0.05_F3_3; Caenorhabditis elegans; RNA-Seq
All reagents except for Triton-X100, PBS-BSA 1%, and inhibitors were prepared the night before and all low retention 1.5mL microcentrifuge tubes were clearly labeled. The bench space and equipment used in this protocol were thoroughly sanitized with 70% ethanol and cleaned with RNaseZAP (AM9780 Fisher) before starting the dissociation. Reagents were prepared using RNase free water (BP2484100 Fisher). The FA lysis buffer was made using the following reagents: 50mM HEPES/NaOH pH 7.5, 1mM EDTA, 0.1% Triton X-100, 150mM NaCl, Protease inhibitor 0.5X (Roche 11697498001), RNase inhibitor 0.2U/μL (Thermo Fisher 10777019), and RNase free water and stored at 4°C or on ice. BSA was prepared to a final concentration of 1% in pH 7.4 1X PBS (AM9624 Thermo Fisher) using RNAse free water and RNAse free PBS. This solution was filtered using a 0.22μm pressure filter (Thermo Scientific 03-377-26, Fisher SLGP033RS). All equipment and reagents were moved to a 4°C cold room and subsequent steps were performed at 4°C.Homogenizers were stored pre-chilled in -20°C when not in use and moved to the 4°C room before starting the extraction. Each wheaton 1.5mL Dounce homogenizers (Z378623-1EA Sigma) was cleaned using 70% ethanol, RNaseZAP, and RNase free water. Homogenizers were rinsed twice with ethanol, twice with RNaseZAP, and 5 times with 1-2mL of RNase free water. The compact 30μL pellet of adult C. elegans was transferred to the dounce homogenizer and 400μL of ice cold FA buffer was used to rinse any remaining worms from the 1.5mL low bind microcentrifuge tube and 1000μL low bind pipette tip and added to the homogenizer. Worms were homogenized with 10 strokes of the dounce homogenizer using a corkscrew motion with a B (tight) pestle. Homogenized worms were transferred to a new low bind 1.5ml microcentrifuge tube and centrifuged at 100g for 1 minute to pellet debris. The supernatant containing the dissociated nuclei was removed using a 1000μL low bind tip and transferred to a fresh low bind 1.5mL microcentrifuge tube labeled pooled nuclei. 300μL of FA buffer was added to the debris remaining in the first microcentrifuge tube and homogenized using 10 strokes in a corkscrew fashion with an Eppendorf Dounce homogenizer. The newly homogenized sample was then centrifuged at 100g for 1 minute to pellet debris. The supernatant containing the newly dissociated nuclei was pooled with the previously dissociated nuclei and the previous steps with the Eppendorf Dounce homogenizer were repeated once more to further homogenize the sample. In total, worms were homogenized with 30 strokes: 10 strokes with the 1.5mL Wheaton Dounce homogenizer and 20 strokes with the Eppendorf dounce homogenizer. Between each homogenization step, debris was pelleted using 100g for 1 minute and the supernatant containing the dissociated nuclei was removed and added to a single 1.5mL microcentrifuge tube labeled pooled nuclei. Dissociated nuclei were removed after each set of 10 homogenization strokes to prevent overdigestion of nuclei. After homogenization, the pooled supernatant containing the dissociated nuclei was centrifuged at 100g for 1 minute to pellet any remaining or accidentally transferred debris. The top 900μL of supernatant containing nuclei was transferred to a clean low bind 1.5mL microcentrifuge tube, being careful not to disturb the debris pellet. These pooled nuclei were pelleted at 500g for 4 minutes. After pelleting, approximately 800μL of FA buffer was removed, being careful not to disrupt the nuclei pellet, and the pelleted nuclei was resuspended with 1000μL of PBS-BSA 1%. The nuclei was again centrifuged at 500g for 4 minutes and 1000μL of the PBS-BSA 1% supernatant was removed. Lastly, the nuclei pellet was resuspended in 750-850μL of PBS-BSA 1% (final volume was determined by examining the size of the nuclei pellet). After resuspension, the nuclei was filtered using a 40μm Flowmi tip filter (BAH136800040-50EA Sigma Aldrich). Filtered nuclei were transferred to a 1.5mL low retention microcentrifuge tube for FACS sorting or 10X sequencing. Library preparation was performed by UCLA Technology Center for Genomics & Bioinformatics. Nuclei were isolated into single droplets and barcoded using the 10X platform. We sequenced using 50bp long paired end reads with the NovaSeq 6000 . 