title: Illumina HiSeq 4000 sequencing; Single-cell analysis of T-cells from mouse neonate thymuses in a wild type control and a placental specific isoform of Igf2 knock-down (igf2-P0)
design: Single-cell analysis of T-cells from mouse neonate thymuses in a wild type control and a placental specific isoform of Igf2 knock-down (igf2-P0)
library_construction_protocol: Thymuses were dissected from postnatal day (PND)6 neonates and homogenised in PBS before passing through a 40 µm filter. Cells were counted and diluted to 200,000 cells / mL in PBS/0.1% BSA for processing according to Drop-Seq protocol (Macosko et al, 2015)  Exon U2 was deleted from the Igf2 locus to prevent expression of the placenta-specific Igf2 transcript, P0. This gene is paternally expressed, so by crossing wild type females with heterozygous males, both wild type and affected neonates grow within the same environment. Single cells were encapsulated in droplets with primer barcoded beads. After RNA binding to the oligo-dT portion of the primer, the droplets were broken to pool beads for reverse transcription. Exonuclease treatment removed unused primers from the beads prior to PCR amplification at up to 8,000 beads / tube (~400 cells/tube). Samples were pooled up to 4,000 cells / sample and purified before determining concentration.  DNA was fragmented and tagged with Illumina adapter and index sequencers using Nextera Sequencing kit and 600 pg DNA/sample. Samples were diluted 1:1 in ddH20 water before double-sided purification with Agencourt Ampure beads using 0.6x beads to remove large fragments and 1x beads twice to remove contaminating primers.
attributes.tag: Experimental Factor: genotype
attributes.value: igf2-p0 heterozygous knockout