GSM3184994
GSM3184994: mock.B.sc65; Mus musculus; RNA-Seq
Single cells were immediately sorted using a FACSaria III (BD Biosciences; device: 48-well plate; precision: single-cell; nozzle: 100 μm) into different wells from a 48-well plate ﬁlled with 4 µl of a hypotonic lysis buffer made of 2U/µL RNase inhibitor and 0.2 % Triton-X-100 in Nuclease-free water. Single-sorted cells were spun down, immediately chilled in a 4°C and stored at −80 °C. After thawing the plates 3 min at room temperature, 0.4 µL of 10X PBS (pH=7.4, Ambion) was added in every well followed by dispensing 4.4 µL of 2X Iodoacetamide (IAA) (Thermo Scientific) to reach a final concentration of 10mM IAA and 50% DMSO. The reaction mixture was incubated for 5 min at 50°C. IAA reaction was quenched with 1.3 µL of 0.1 M DTT (final concentration of 15 mM) for 5 min at room temperature. ERCC spike-in control (Thermo Fisher, mix 1, dilution 1:2 millions) was added in every well (except the negative control). RNA was isolated before Reverse Transcription and PCR using a 1:1 volume of RNA XP magnetic beads (Beckman Coulter). After 10 min incubation at room temperature, the beads were washed twice with 50 µl 80% freshly-prepared EtOH on the magnetic stand and the dried beads pellets were resuspended in 4 µL elution buffer (RNAse inhibitor 1/200 Takara, CDS primer 0.5 µL in RNAse free water) immediately followed by an incubation at 72°C for 3 min. All the following experimental steps were performed using the Smart-seq v4 low input kit (Takara) with one-quarter of the recommended reagent volumes. The PCR amplification was performed according to the manual using 24 cycles. Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent).0.5 ng of every library was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using one-quarter of the recommended reagent volumes, 10 min for tagmentation at 55 °C and 1 min extension time during PCR for multiplexing. After PCR, the libraries were purified using AMPure XP beads and resuspended in 15 µl of resuspension buffer. single-cell SLAM-Seq (Smart-Seq v4 kit) bulk SLAM-Seq (TruSeq kit)
GEO
GSM3184994
GEO Accession
GSM3184994