Chromium single cell 3 library gel bead kit v3

Organoids were cultured to 120 days, then washed in PBS and dissociated using the gentleMACS Dissociator (Miltenyi Biotec) in program NTDK1 using the enzyme mix: Trypsin (Sigma Aldrich)/Accutase (Sigma Aldrich; 1:1) containing 10 U/ml DNaseI (Thermo Fisher Scientific). The washed cell suspension was passed through a 70 μm cell strainer.
In the newly generated datasets, we pooled cells from samples from four different genotypes and were combined in a lane (other cell lines used for other purposes). We additionally used sample barcoding using lipid‐anchor barcoding following instructions as in (McGinnis et al, 2019 (link)) with reagents kindly provided by the authors, but we relied on SNP‐based cell line demultiplexing as described in (Kang et al, 2018 (link); described in the following section) and sample barcoding was not used.
Cells were counted and the suspension was loaded onto a Chromium Single Cell 3′ B Chip (10× Genomics, PN‐1000073) and processed through the Chromium controller to generate single‐cell GEMs (Gel Beads in Emulsion). scRNA‐seq libraries were prepared with the Chromium Single Cell 3′ Library & Gel Bead Kit v.3 (10× Genomics, PN‐1000075). Ready 10× libraries were sequenced paired end (R1:28, R2: 89 cycles) on NovaSeq (Illumina).

Hippocampal neural stem/progenitor cells (NPCs) were isolated by microdissection from E17 day embryos (offspring of male Kmt2d^+/βgeo and female C57Bl/6J) and cultured on Matrigel as described in [24 ]. Cells were maintained in an undifferentiated state via supplementation with growth factors (EGF, FGF2) in Neurobasal media. In a prior publication, we have demonstrated that the Kmt2d^+/βgeo cells exhibit defects in proliferation [24 ]. Replicate cultures from both genotypes were collected at the undifferentiated state (day 0) and then 2, 4, and 8 days after growth factor removal to induce neuronal differentiation. Cells were collected via trypsinization and pellets were washed and resuspended in Neurobasal media. scRNA-seq libraries were created with a Chromium Single-Cell 3’ library & Gel Bead Kit v3 (10x Genomics) according to the manufacturer’s protocol. Approximately 10,000 cells were targeted for each library. Only cells from day 0 are analyzed here.

Libraries for Single cell RNA seq were generated using Chromium single cell 3′ library & Gel Bead Kit v3 (PN-1000092, 10X Genomics), Chromium Single Cell B Chip kit (PN-1000074, 10X Genomics). Briefly, cells were mixed with reverse transcription (RT) reaction reagent and loaded onto the B chip aiming for 6000 captured cells per a channel. After RT reaction, gel bead-in-emulsions were transferred to tubes and performed RT reaction using thermal cycler (C1000 Touch, Bio-Rad, RRID:SCR_019688). cDNA was purified using Dynabead MyOne SILANE (Thermofisher) and further amplified. Subsequent steps for library construction were performed by the manufacturer’s instruction provided. Quality of the amplified cDNA and final libraries were monitored by Bioanalyzer (Agilent, RRID:SCR_019715). Libraries were sequenced with a 2 × 100 bp paired-end protocol on a Novaseq S4 platform from Illumina to generate minimum 20,000 read pairs per cell.

Multiplexed scRNA-Seq profling involved iNs from 4 distinct donors per library. Neuronal networks were dissociated 49 days after differentiation with papain (10 units/ml) and DNAse (100 units/ml) diluted in DMEM/F12 for 10 min at 37 °C to facilitate generation of a single cell suspension. Afterwards, the network was mechanically sheared with a P1000 pipette tip. DMEM/F12 supplemented with 20% FCS was added for centrifugation at 400 g for 5 min at RT. Cells were resuspended in DMEM/F12 supplemented with 1% BSA and 1x RC and pelleted for a final resuspension in 1 % BSA in DPBS supplemented with RNAse Inhibitor, 1x RC, 3 mM Mg(Ac)₂, and 5 mM CaCl₂. After removal of network debris with filters, cells were counted. 12,000 cells (3,000 per donor) were loaded on the 10X Chromium machine (10X Genomics). scRNASeq libraries were prepared following manufacturer’s instructions of the Chromium^™ Single Cell 3’ Library & Gel Bead Kit v3 (10X Genomics). The scRNA-Seq library was subsequently sequenced/processsed on a NovaSeq 6000 S1 flowcell.

Single cell suspensions were isolated by Ficoll-Paque Plus (17144002, GE Healthcare Life Sciences, Pittsburgh, PA). Chromium^TM Single Cell 3′ Reagent Kits v2 (PN-120237, 10× GENOMICS) were used to perform single-cell separation, cDNA amplification and library construction following the manufacturer’s guidelines. A high-sensitivity dsDNA Qubit kit was used to quantify the cDNA concentration. The HS DNA Bioanalyzer for cDNA (or lower concentrated libraries) or the DNA 1000 Bioanalyzer was used to measure the concentration of the libraries. The barcoded library at the concentration of 1.6 pm was sequenced on the NextSeq500 v2.5, High Output flow cell using a 26 × 124 sequencing run format with 8 bp index (read 1).
The cellular suspensions were loaded on a 10× Chromium Single Cell Controller to generate single-cell Gel Bead-in-Emulsions (GEMs). The scRNA-Seq libraries were constructed using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (P/N 1000092, 10x Genomics). The barcoded libraries were quantified and then sequenced using the HiSeq4000 System (Illumina, San Diego, CA).