GSM3039459: Trunk 1.2; Schmidtea mediterranea; RNA-Seq
Planarian body sections were cut into small fragments (<1mm) and transferred to a 50 mL tube with CMFB (400mg/L NaH2PO4, 800mg/L NaCl, 1200 mg/L KCl, 800 mg/L NaHCO3, 240 mg/L glucose, 1% BSA, 15mM HEPES, pH7.3). Volume was brought to 45 mL with CMFB, to which 5 mL collagenase (1 mg/ml; Sigma-aldrich, C0130) was added. Suspension was generated through agitation of the mixture for 5-10 minutes using pipettes. Cell suspension was passed through a 40μm filter and centrifuged at 1250 rpm for 5 minutes. The supernatant was removed and cells were resuspended in 1 mL of CMFB. Single cell suspensions were stained with Hoechst 33342 (40 μl/ml, Invitrogen) and propidium iodide (3 μl/ml, Sigma-aldrich). Living cells were sorted by flow cytometry into 5 mL tubes. In seven samples, cells with 4C DNA content as determined by high Hoechst signal were gated out. Following FACS, cells were isolated in droplets with barcoded beads using Drop-seq (http://mccarrolllab.com/download/905/). Briefly, cells were lysed within droplets, capturing polyA RNA onto barcoded beads. Barcoded beads were isolated and used to generate cDNA. Beads with attached cDNA were exonuclease treated and cDNA was amplified by PCR. Pairs of PCR reactions were purified using 0.6X AMPure XP beads (Agencourt).  Prior to Drop-seq runs, cultured cells were grown to confluence. Cells were washed once with 1X PBS. NIH/3T3 cells were then treated with TrypLE (Invitrogen) for five minutes and quenched and resuspended in an equal volume of growth medium. 293 T cells were resuspended in 1X PBS by manual dissociation. Cells were spun down at 300xg for five minutes and resuspended in 1X PBS + 0.01% BSA. Cells were spun down again at 300xg for three minutes and resuspended in 1 mL 1X PBS. Cells were passed through a 40μm filter and counted. 1X PBS + 0.01% BSA was used to generate input for Drop-seq runs (http://mccarrolllab.com/download/905/). A single library was generated for each Drop-seq run using 600pg purified, amplified cDNA using Nextera XT (Illumina). Libraries were purified by two rounds of AMPure XP purification (0.6X, 1X) (http://mccarrolllab.com/download/905/).
GEO Accession
GSM3039459