GSM2301372: C. angaria 2-cell rep 1; Caenorhabditis angaria; RNA-Seq
Pellets of embryos were resuspended in 2 mL of Ringer’s solution (made with DEPC water) + 0.01% tween 20. DEPC was used in the Ringer’s solution to limit RNase contamination, and tween 20 was used to prevent embryos from sticking to any surfaces. Resuspended embryos were passed through at 40 μm mesh filter into a 60mm x 15mm petri dish to remove debris. Enough Ringer’s solution + 0.01% tween 20 was added to coat the bottom of the petri dish and reduce the density of the embryos so that they could easily be collected. Embryos were visualized in the dish using an EVOS inverted microscope, and single embryos were imaged and collected in 1.5 μL using a micropipette. If more than one embryo was collected, embryos were diluted further by pipetting them into 20 μL Ringer’s solution + 0.01% tween 20 on a clean slide that was pretreated with RNase ZAP or 70% ethanol. Single embryos were collected in 1.5 μL into PCR tube strip, and 2 μL of lysis buffer (18 μL 0.3% Triton-X 100 + 2 μL RNase inhibitor SIGMA), 1 μL of oligo-dT primer, and 1 μL of dNTP mix were added to each embryo. Embryos were heated to reverse secondary structure of RNA, reverse transcribed and PCR amplified according to the Smart-seq2 protocol by Picelli (Picelli et al. 2014) (Supp. Figure 1). All embryos, regardless of embryonic stage, were amplified for 18 cycles through PCR. PCR primers were cleaned up from the embryo samples by adding a 1:1 ratio of Ampure XP beads to sample, which were both equilibrated to room temperature, incubated for 8 min, placed on a magnet, and washed with 200 μL of 80% ethanol 3 times. Beads were dried at room temperature for approximately 5 min (until the beads cracked), after which, 17.5 μL of EB was added and incubated off the magnet for 3 min. Samples were placed back on the magnet, and 15 μL of cDNA was collected for each sample. Sample cDNA concentration was quantified using the Qubit fluorometer and bioanalyzed using the Agilent 2100 Bioanalyzer to check the cDNA quality. For library preparation, 20 ng of cDNA from each sample was prepared using the regular Nextera tagmentation protocol (Gertz et al. 2012). The protocol reagents were scaled down, so that 2 μL of transposase, 10 μL of buffer, and 8 μL of cDNA (20 ng total) were used yielding a total volume of 20 μL. Transposase was cleaned up from the tagmented DNA using the QIAGEN columns as follows. Three volumes of buffer PM was added to each sample, placed into a QIAGEN spin column and spun at 13,000 RPM for 1 min. Flow-through was removed, 750 μL of buffer PE (prepared with ethanol) was added, and samples were spun at 13,000 RPM for 1 min. Flow-through was removed, and samples were spun again at the previous settings to dry the columns. Columns were placed into new clean collection tubes, and 30 μL of EB prewarmed to 55oC was added to the center of each column and incubated for 1 min before spinning down at the same settings. In a PCR tube, 30 μL of sample, 35 μL of Phusion high fidelity master mix, 2.5 μL 25 μM Nextera adapter ID, and 2.5 μL 25 μM Nextera adapter Ad_noMX were combined and mixed well with a pipette. Samples were spun down quickly, and amplified for 6 cycles using the PCR program with the following settings: 72 °C for 5 min, 98 °C for 30 s, [98 °C for 10 s, 63 °C for 30 s, 72 °C for 1 min] for 6 cycles, 72 °C for 5 min, and hold at 4 °C. PCR amplified libraries were cleaned up using a 1:1 ratio of Ampure XP beads to sample, and prepared in the same way as the bead cleanup above, except that 30 μL of EB was added to the beads to resuspend the library sample, which was then collected in 27.5 μL after 2 min. Sample library fragments were between 200-600 bps with an average size of 360 bps after the Nextera tagmentation protocol. Samples were sequenced as paired-end 43 bp on the Illumina NextSeq 500 to an average depth of ~10 million reads.