GSM6600115: SJG26_NT_plate3_mRNA; Homo sapiens; RNA-Seq
The RAID staining was performed as previously described (Gerlach et al., 2019). Briefly, cultured cells were dissociated, followed by fixation with 2.5 mM DSP and 2.5 mM SPDP in sodium phosphate buffered saline (pH 8.4) for 45 minutes at RT. The fixation was quenched by adding 100 mM Tris-HCl pH 7.5 and 150 mM NaCl for 10 minutes. Subsequently, the cells were blocked and permeabilized for 30 min at RT in a mix of 0.5x PFBB (protein free blocking buffer, Thermo Fisher Scientific) in PBS supplemented with 10 µg/ml tRNA (Roche), 0.5 U/µl RNAsin Plus (Promega) and 0.1% Triton X-100. The panel of ssDNA-labelled antibodies was mixed with 2 U/µl RNAsin Plus (Promega) and 0.1% Triton X-100 in PBS:PFBB (1:1) and used to stain the cells. The staining was performed overnight at 4°C on a rotating mixer. To remove unbound antibodies, the cells were washed four times after the staining with a wash-buffer (10% PFBB and 0.05% Triton X-100 in PBS). Subsequently, the cells were washed two more times in 10% PFBB in PBS, followed by single cell sorting (BD FACS Aria) into 384-well plates containing CEL-seq2 primers. The library preparation is based on the previously published protocol (Gerlach et al., 2019), with some adaptations. ERCC spike-in (1:100,000 dilution, Thermo Fisher Scientific) was added to the reverse crosslinking mix. The transcriptome and antibody barcodes were separated by performing a double bead-purification after first strand synthesis and pooling. The RNAs were selected using a 0.6x Ampure XP bead ratio (Beckman Coulter) purification, while the antibody barcodes were selected through 2.0x Ampure XP bead ratio purification. The RNAs were subsequently subjected to second strand synthesis and in vitro transcription as previously described (Gerlach et al., 2019). The antibody barcode part was first linearly amplified using FWD pre-amplification primer (5' CACGACGCTCTTCCGATCT 3') for 10 cycles followed by addition of T7 primer (5' GCCGGTAATACGACTCACTATAGGG 3') for 2 cycles to generate dsDNA. The dsDNA generated was then subjected to in vitro transcription using Megascript T7 transcription kit (Thermo Fisher Scientific). The amplified RNA from the transcriptome part and antibody barcode part was subjected to reverse transcription primed with random octamer with FWD handle (5' CACGACGCTCTTCCGATCTNNNNNNNN 3') and FWD pre-amplification primer (5' CACGACGCTCTTCCGATCT 3'), respectively. Subsequently, the cDNA was amplified followed by Illumina sequencing index addition as previously described (Gerlach et al., 2019). The sequencing was performed with Illumina Nextseq 500.