title: GSM5369498: vasadrop_E6.5-2_i2; Mus musculus; RNA-Seq
library_construction_protocol: vasaplate: Single cells were sorted by FACS into 384-well plates containing RT-primers and oil. Reagents were dispensed with a robot and plates incubated in a thermocycles after each dispensing. Briefly; 1) Lysis/fragmentation mix, 2) Poly(A)-tailing/end repair mix, 3) Reverse transcription mix, 4) Second strand synthesis mix. After second strand synthesis, wells were pooled and material was amplified with IVT. Amplified material was depleted of rRNA, second adapter was ligated and aRNA converted to cDNA. Full Illumina handles were added by PCR, and samples were sequenced. vasadrop: Single-cells were co-encapsulated with barcoded inDrop v3 beads with with a lysis and RNA fragmentation buffer. The droplets were then incubated and further injected with a RNA repair and poly(A) tailing mix using a pico-injector. After incubation, the droplets were the injected with a reverse transcription mix and incubated. The. Droplets were then de-emulsified and processed similarly to the VASA-plate workflow. cell dissociation: HEK293T were cultured in DMEM, dissociated with Trypsin-EDTA and washed with ice-cold PBS. Mouse ES cells were cultured in 2i/LIF conditions, dissociated with Accutase and washed with ice-cold PBS. Mouse embryos from E8.5 and E9.5 were cut into pieces under the microscope before collection in a tube, embryos from stage E6.5 and E7.5 were not cut. TrypLE Express was used for cell dissociation and washed with PBS supplemented with 0.4% BSA. Illumina TruSeq adapaters