title: GSM8833955: Pooled mouse EBs at day 21 with CRISPR perturbations from a pilot experiment; Mus musculus; RNA-Seq
library_name: GSM8833955
library_construction_protocol: To obtain a single-cell suspension for downstream single-cell experiments, EBs at a given time point, typically at day 21 for the mosaic perturbation screens or at day 7, 14, or 21 for the wildtype time series experiment, were collected into 50 mL conical tubes at a density of three plates per tube and allowed to settle by gravity. The old CA medium was carefully removed and replaced with PBS (-/-) for two sequential washes, allowing EBs to settle by gravity after each wash. After removing the final wash, 3 mL of 0.25% trypsin was added to the tube, and the EBs were incubated at 37C for 5 min with gentle agitation. To achieve a uniform single-cell suspension, the trypsinized EBs were carefully pipetted up and down using a P1000 pipette followed by quenching with CA medium. The cell suspension was passed through a 40 um cell strainer, centrifuged at 300g for 5 min, and the cell pellet resuspended to a final concentration of 2 million cells / mL. FAC sorting was performed to enrich for a clean single-cell population, after which the cells were centrifuged at 400g for 5 min and resuspended in cold PBS containing 0.04% BSA at a concentration of approximately 1200 cells/uL. The resulting single-cell suspension was then processed following the 10x Genomics protocol as per the manufacturer's instructions, loading 16K cells per lane for an estimated recovery of 10K cells.  For dissociating monoclonal EBs into a single-cell suspension, CA media was removed from each 2 mL tube, and 750 µL of 0.25% trypsin was added. Tubes were incubated at 37°C on a thermomixer set to 1100 rpm, with dissociation monitored under an epifluorescence microscope at 5-minute intervals. At the 10-minute mark, gentle trituration with a P1000 pipette was performed to aid dissociation, with additional trypsin added if necessary. Complete dissociation was typically achieved within 20 minutes. Trypsin was inactivated by adding CA media, and the cell suspension was further diluted before being passed through a 45 µm strainer into a 50 mL conical tube containing 10 mL of CA media. Cells were counted using a hemocytometer, transferred to a 15 mL conical tube for ease of handling, and centrifuged at 400g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in PBS + 0.04% BSA. The initial cell concentration was adjusted to 2400 cells/µL. A second cell count was performed, and the suspension was further diluted to a final concentration of 1200 cells/µL. Single-cell suspensions from each condition were then processed using the 10X Genomics High Throughput (HT) kit, with cells loaded across 16 lanes. Wild-Type Time Series: Libraries were prepared using the 10x Genomics Single Cell 3' v2 kit. Three time points (days 7, 14, and 21) were sequenced, with one lane per time point, for a total of three lanes. Final libraries were sequenced on a NextSeq 500 using a 75-cycle high-output kit (R1: 26 bp, I1: 8 bp, I2: 0 bp, R2: 57 bp).  Pilot Screen: Libraries were prepared using the 10x Genomics Single Cell 3' v3 kit. Replicate 1: Six lanes were sequenced: two lanes for CRISPRcut sorted, two lanes for CRISPRi sorted, one lane for CRISPRcut unsorted, and one lane for CRISPRi unsorted. Final libraries were sequenced on a NextSeq 500 using two 75-cycle high-output kits (R1: 26 bp, I1: 8 bp, I2: 0 bp, R2: 57 bp). Replicate 2: Six lanes were sequenced: three lanes for CRISPRcut and three lanes for CRISPRi. Final libraries were sequenced on a NextSeq 500 using two 75-cycle high-output kits (R1: 28 bp, I1: 8 bp, I2: 0 bp, R2: 55 bp).  All TF Screen: Libraries were prepared using the 10x Genomics Single Cell 3' v3 kit. A total of 32 lanes were sequenced for CRISPRcut. Final libraries were sequenced by the Northwest Genomics Center on a NovaSeq 6000 using one S2 flow cell and one S4 flow cell (R1: 28 bp, R2: 80 bp, I1: 10 bp).  125TF Screen: Libraries were prepared using the 10x Genomics Single Cell 3' v3 kit. A total of 32 lanes were sequenced for CRISPRcut. Final libraries were sequenced by the Northwest Genomics Center using both a NovaSeq 6000 with one S4 flow cell (R1: 28 bp, I1: 10 bp, I2: 0 bp, R2: 91 bp) and a NextSeq 2000 with a P3 100-cycle kit (R1: 28 bp, I1: 8 bp, I2: 0 bp, R2: 91 bp).  piggyFlex and monoclonal EB validation: Sequencing was performed using four lanes of the 10X Genomics Single Cell 3' v3 kit, with two lanes allocated for replicate 1 and two lanes for replicate 2. Final libraries, capturing both gene expression and directly captured gRNAs, were sequenced on a NextSeq 2000 P2 100-cycle kit (R1: 28 bp, I1: 8 bp, I2: 0 bp, R2: 91 bp). Organoid barcode (BC) transcripts were separately sequenced using a NextSeq High 75-cycle kit (R1: 28 bp, I1: 10 bp, I2: 0 bp, R2: 53 bp).  TF screen in monoclonal EBs: Sequencing for the TF screen in monoclonal EBs was performed using 16 lanes of the 10X Genomics Single Cell 3' HT Kit v3.1, with 12 lanes dedicated to conditions 1–10 and 4 lanes for condition 11. Final libraries, capturing gene expression, directly captured gRNAs, and organoid BCs, were sequenced on a NextSeq 2000 P3 100-cycle kit (R1: 28 bp, I1: 10 bp, I2: 10 bp, R2: 90 bp).
identifiers.namespace: GEO
identifiers.id: GSM8833955_r1