The organizer as a cooperative of signalling cells for neural induction
The organizer as a cooperative of signalling cells for neural induction
For collection of single cells for single cell re-grafts and scRNA-seq, the cultured embryo was first submerged in Tyrode's solution (for re-grafts) or sterile molecular grade PBS with 0.1% glucose (for scRNA-seq). The node was then divided into anterior, middle and posterior sub-regions of equal rostro-caudal length. Each of these regions containing GFP-positive cells was cut out, in turn, and placed in a drop of non-enzymatic dissociation medium (Sigma, # C5914-100ML), kept over ice. Each piece was washed twice in drops (~30 µl) of this dissociation medium while over ice. To help with dissociation, after ~5 min, the tissue was gently aspirated up and down using a micropipet (made from a pulled 50 µl calibrated micropipet (Drummond Scientific, Cat 2-000-050) attached to an aspirator tube). The micropipet was broken at the tip to have a diameter just narrower than the width of the tissue piece. Once the piece of tissue was fragmented, a capillary with a narrower tip was used for further dissociation to single cells in suspension. GFP-positive cells were identified by fluorescence under a dissection microscope (x70 magnification) and were individually aspirated using a micropipet. The cell was transferred into a drop of Tyrode's (for re-grafts) or of sterile molecular grade PBS (scRNA-seq) to replace the dissociation medium and to verify that there was only a single GFP-positive cell. Once verified, the cell was transferred (using a fresh pulled micropipet) to the second host (for re-grafts) or into a 200 µl tube containing 5 µl of lysis buffer and 5% RNase inhibitor (for scRNA-seq) (lysis buffer and RNase inhibitor from SMART-Seq v4 Ultra Low Input RNA Kit, Takara, # 634892). Once the dissociation process began, cells were collected for ~20 min, after which time any remaining dissociated tissue was discarded, and a new tissue piece taken from the embryo. The SMART-Seq v4 Ultra Low Input RNA Kit (Takara, # 634892) targeting mRNA, was used for preparing the single cells for sequencing. Amplified cDNA was purified using AMPure magnetic purification beads (Agencourt AMPure XP, Beckman Coulter # A63880). The DNA concentration of purified cDNA was checked by Qubit (dsDNA HS Assay Kit, Thermofisher # Q33230). All samples yielding at least 5 ng of cDNA were sheared by sonication (Covaris, S220/E220 focused-ultrasonicator, settings set to: 10% duty factor, 200 cycles per burst, 120 second treatment time, 175W peak incident power) to obtain ~500bp fragments for library preparation. The ThruPLEX DNA-seq, Dual Index Kit (Takara, # R400406) was used to construct dual indexed libraries for each sample. Libraries were individually purified, using AMPure magnetic purification beads (Agencourt AMPure XP, Beckman Coulter # A63880).