GSM6857606: Smarcc1-mut; Mus musculus; RNA-Seq
Organoids were isolated on day 5 after passaging and dissociated into single cells using TrypLE incubation. 20,000 live cells were sorted into 10 mL of ultra clean 0.8% BSA/PBS For scRNA-seq of mutant FEnS (Smarca4mut, Smarcc1mut and Scramble-ctrl) TotalSeq™ hashtag antibodies (A0301-A0305; Biolegend) were used to multiplex samples. Cells isolated from two individual organoid lines from each perturbation were sequenced. Cells were isolated on day 5 after passaging and dissociated to single cells. Each sample was incubated with 0.5 mg of unique hashtag antibody for 20 minutes on ice. Before sorting, cells were incubated with DAPI (0.2 mg/mL) for 10 minutes on ice. 10,000-15,000 events/cells from each sample were sorted on an ARIAIII sorter (BD Biosciences) and pooled directly into four tubes. Single cell libraries were prepared using 10X Genomics v3 Chemistry as described above with implementation of hashtag libraries for demultiplexing. An additive primer (HTO primer) was added in the step for cDNA amplification. The hashtag-cDNA and endogenous cDNA were separated after cDNA amplification using SPRIselect beads (Beckman Coulter) for size separation. Here hashtag-cDNA was isolated in the supernatant containing smaller DNA fragments and collected to generate the hashtag-library following the protocol from Stoeckius et al and CiteSeq (https://cite-seq.com). Briefly, the supernatant was cleaned using SPRIselect beads (Beckman Coulter) and 30 ng cDNA was used as input for PCR amplification (20 cycles). Illumina-indexes for sequencing was subsequently added using Kapa HiFi HotStart Ready Mix (Roche Diagnostics). For the endogenous cDNA library, the 10X protocol was followed. The dual index kit set AA (10X genomics) was used for sample indexing and 11-12 cycles were used during the final amplification step. The four hashtag cDNA- and four endogenous cDNA libraries were diluted to 2 nM and combined into two pooled libraries for sequencing (5% hashtag + 95 % endogenous cDNA). The two final libraries for sequencing were further diluted to 1.7 pM before being sequenced with Illumina High Output 150 cycles kit on an Illumina NextSeq500 sequencer.