title: GSM9239908: mix of four wild-type and four Uhrf1 knockout E8.5 embryos, sublibrary 4, GEX; Mus musculus; RNA-Seq
library_construction_protocol: Uhrf1​+/−​ male and female mice were naturally mated and checked every morning for the appearance of a vaginal plug, the presence of a plug determined the time point E0.5. E8.5 embryos were dissected from the uterus in PBS/FBS and extra-embryonic tissue was removed.The embryos were washed in PBS without Ca2+/Mg2+ and each was put in a 3 cm dish with 1 ml TrypLE, chopped into small pieces, and incubated at 37°C for 20 min. 300 μl serum was added and pipetted up and down until tissue was dissociated in single cells. Briefly, cells were fixed and permeabilized. For joint measurements of single cell chromatin accessibility, DNA methylation, and expression, cells were first transposed by Tn5 transposase to mark regions of open chromatin. The mRNA was reverse transcribed using a poly(T) primer containing a unique molecular identifier (UMI) and a random primer. Both primers contained a biotin tag. Permeabilized cells were distributed in a 96-well plate to hybridize well-specific barcoded oligonucleotides to transposed chromatin fragments and poly(T) cDNA. Hybridization was repeated three times to expand the barcoding space and ligate cell barcodes to cDNA and chromatin fragments. Reverse crosslinking was performed to release barcoded molecules. cDNA was separated from chromatin using streptavidin beads, and each library was prepared separately for sequencing. Chromatin DNA was purified, gap-filled and oxidized using mTET1, and deaminated with 1 M pyridine borane before amplification. Both DNA and RNA libraries were quantified with KAPA Library Quantification Kit and pooled to 4 nM for sequencing. Libraries were sequenced on the Illumina Nova-seq platform with 150 PE reads (Read 1: 150 cycles, Index 1: 8 cycles, Index 2: 8 cycles, Read 2: 150 cycles). Libraries were sequenced on the Illumina NovaSeq platform, aiming for a minimum coverage of 50,000 reads per single cell.