Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: A working lysis buffer solution was prepared by adding 1 µL of the MNase (NEB) [1:50 dilution] per 5 µL lysis buffer. To lyse the mouse samples, 6 µL of working lysis buffer was added directly to the frozen cell-containing droplet. Digestion was immediately performed for 30 minutes at 37 °C in a thermal cycler with a heated lid. 1 µL EGTA was added to a final concentration of 10 mM to inhibit further digestion. Samples were placed on ice until processing through Ribo-ITP. For 100-cell human K562 cells and all mouse samples, Ribo-ITP outputs were immediately processed through the D-Plex Small RNA-seq Kit (Diagenode C05030001) with minor modifications as detailed here. The dephosphorylation reaction was supplemented with 0.5 µL T4 PNK (NEB) and the reaction was incubated for 25 minutes. For reverse transcription, the template switching oligo (TSO) was diluted 1:2 in nuclease-free water. All 100-cell human samples and three of the MII-stage oocytes were processed using the single index (SI) module; while the other mouse samples were processed using the unique dual index (UDI) module. Half of the cDNA was amplified for 17 PCR cycles and a 1:4 dilution of the resulting library was assessed by Agilent Bioanalyzer High Sensitivity DNA kit. The concentrations of the target peaks were used to pool samples with approximately equimolar representation. AMPure XP bead cleanup (1.8x) was performed followed by size-selection using 3% agarose, dye-free gel cassettes with internal standards (Sage Science BDQ3010) on the BluePippin platform. Tight parameter settings of 173-207 bp range were used for samples prepared with the SDI module. Tight parameter settings of 183-217 bp range were used for samples prepared with the UDI module. Samples were sequenced on a Illumina NovaSeq 6000.