GSM5859164: CD1 Drop-seq rep1; Mus musculus; RNA-Seq
pSHFs and HT were microdissected from ~80 embryos and pooled for each sample. Tissue was dissociated in TrypLE for 5 minutes at room temperature with shaking at 1000 RPM, before quenching with 10% FBS. Cells were centrifuged at 800 x g and washed with 0.01% BSA in PBS. Two single cell RNA-seq libraries were generated using the standard Drop-seq protocol (Macosko et al., 2015) with minor modifications. Briefly, cells were filtered using a 40 µm mesh filter (Nalgene, USA) and suspended in 1x PBS and 0.01% BSA at a concentration 120,000/mL. Custom-synthesized Drop-seq barcode (BC) beads (Chemgenes, USA) at 100,000/mL were likewise filtered trough 100 µm mesh filter and suspended in Drop-seq Lysis Buffer. Using 3 mL syringes for each, the solutions were co-encapsulated in monodisperse droplets of 125 µm in an inert carrier oil (BioRad Evagreen) using microfluidic device fabricated in-house. The cells underwent lysis in the microfluidic droplets and the mRNA were captured onto the BC beads via polydT oligoes affixed to the BC beads. The droplets were then broken to retrieve the BC beads with mRNA hybridized onto them in high salt solution that stabilized the BC oligo+mRNA complexes. The beads are thoroughly washed and reverse transcription is performed to convert the BC+mRNA complex into barcoded cDNA. An Exonuclease I digestion is performed to digest any barcodes that are unbound to mRNA. PCR of 12-15 cycles is performed to amplify the barcoded single cell library, quantified and the quality of cDNA library is assessed. Sequencing libraries are prepared using Illumina Nextera kit with sample-specific indices added that allow de-multiplexing each sample from sequence data. The samples were sequenced on the Illumina NextSeq platform using a 75 cycle kit, with a custom Read1 primer (Macosko et al., 2015). The two biological replicates were processed on different days. Drop-seq