| 生长方案: |
To obtain a broad range of embryo ages, including early stages, roughly synchronized C. elegans adults (N2 strain) were obtained by releasing embryos with standard hypochlorite treatment and letting the L1 larvae hatch and undergo growth arrest on unseeded plates. Starved L1s were transferred to NGM plates seeded with E. coli OP50 bacteria. Embryos were released from these synchronized young adults using hypochlorite treatment followed by three washes with L15-10 media. To generate cell suspensions, embryos were then treated with 0.5mg/ml chitinase at room temperature until the shells were dissolved (30-40 minutes at room temperature (~22 degrees C)) followed by dissociation of the cells using a 3ml syringe fitted with 21 1/2 gauge needle until >80% of embryos were disrupted. The cell suspension was then passed through a 10µM filter, washed in phosphate buffered saline (PBS) and finally resuspended in PBS. An estimated 14,000 cells were loaded immediately on a 10X Chromium instrument. The trypan blue negative viable cell count was estimated using a hemocytometer and was >84% for all samples. To sample later stages more deeply, more tightly synchronized embryo populations (used for the 300-minute, 400-minute, and 500-minute time series shown in Fig. 1B) were obtained through two cycles of bleaching adult worms (strain VC2010, a strain derived from N2 that has been completely sequenced). On the first round of synchronization, populations of mixed stage embryos recovered by hypochlorite treatment of mixed populations were hatched overnight in egg buffer (118 mM NaCl, 48 mM KCl, 3 mM CaCl2, 3 mM MgCl2, 5 mM HEPES pH 7.2) with gentle shaking. The hatched L1s were plated onto 150 mm peptone rich NGM plates seeded with E. coli NA22 at no more than 100,000 worms per plate. When worms reached the adult stage, the number of embryos inside the adults was monitored until most had about 4 embryos on each gonad arm. The adult worms were collected and treated with hypochlorite to release embryos. The embryos were again allowed to hatch in the absence of food at 20 °C for 12 hours yielding a more tightly synchronized population of L1 worms. Around 250,000 L1 larvae were plated onto four 100 mm petri plates seeded with NA22 bacteria and allowed to develop at 20 °C. As the worms reached the young adult stage, the population was closely monitored. When about 20-30% of the adults had a single in either arm of the gonad, worms were subjected to hypochlorite treatment. The time hypochlorite was added to the worms was considered t=0 (see Warner et al. in press, for typical age distributions). The capture time was taken as when the cells were loaded onto the 10x Chromium capture. The embryos were allowed to develop in egg buffer until one hour prior to capture time. The embryos were collected by centrifugation, resuspended in 0.5 ml egg buffer and 1 ml chitinase (1 U/ml) and transferred to 30 mm petri dishes. The degradation of eggshell was monitored; after ~20 min (about half the eggs had lost the shell), the suspension was transferred to a 15ml falcon tube and centrifuged at 200 g for 5 min. The chitinase solution was aspirated; a solution of 200 ul pronase (15mg/ml) together with 0.5 ml egg buffer was added to the embryo pellet. The vitelline membrane was disrupted and the cells released by repeated passage through 21G1 ¼ needle attached to a 1 ml syringe. When sufficient single cells were observed the reaction was stopped by adding 1ml of egg buffer containing 1% BSA. Cells were separated from intact embryos by centrifuging the pronase treated embryos at 150 g for 5 min at 4°C. The supernatant was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 500 g for 5 min at 4°C. The cell pellet was washed twice with egg-buffer containing 1% BSA. |
| 提取方案: |
Single cell capture and library preparation followed 10X Genomics published protocols. For each channel, 14,000 C elegans cells were mixed with reverse transcriptase reaction solution and loaded immediately onto the capture chip to minimize the time C. elegans cells spent in reverse transcription cocktail. The exception was the first 500 minute sample, when 14,000, 4,666, and 1,555 cells were loaded on each channel. |
