title: GSM3764784: larva1_rep1; Ciona intestinalis; RNA-Seq
library_construction_protocol: For each sample, 100 to 500 morphologically normal embryos were transferred into tubes pre-coated with 5% BSA in Ca2+-free artificial sea water (Ca2+-free ASW, 10mM KCl, 40mM MgCl2, 15mM MgSO4, 435 mM NaCl, 2.5mM NaHCO3, 7mM Tris base, 13mM Tris-HCl). Embryos were immediately dissociated with 0.5 to 1% trypsin in Ca2+-free ASW with 5 mM EGTA (ASW-EGTA) for 2min (gastrula and neurula stages) to 10min (tailbud stages). Embryos were pipetted 5min on ice to complete dissociation of individual cells.  Then, the digestion was inhibited with 0.2% BSA in Ca2+-free ASW or quenched by 20% FBS. Cells were collected by centrifugation at 4°C at 500g for 2 to 5min and then resuspended in ice-cold Ca2+-free ASW containing 0.5% BSA. For the swimming larva stage, the embryos were either homogenized (H100, Waverly) and dissociated using 1% trypsin or dissociated with 1% trypsin, 1mg/ml collagenase, 0.5% pronase and 0.5mg/ml cellulase in ASW EGTA. Once dissociated, the enzymes were inhibited by 20% FBS and 2mg/ml Glycine. For all the samples, cells were lysed, cDNAs were barcoded and amplified with Chromium Single Cell 3' Library and Gel Bead Kit v2 (10x Genomics, CA) following the instructions of the manufacturer. Illumina sequencing libraries were prepared from the cDNA samples using the Nextera DNA library prep kit (Illumina, CA). All the libraries were sequenced on Illumina HiSeq 2500 Rapid flowcells (Illumina Inc., CA) with paired-end 26nt + 125nt reads following standard Illumina protocols.