GSM7787274: MUC22809 CM-OXT-202-23-F; Mus musculus; RNA-Seq
The single-nucleus RNA-seq2 methodology was used to capture a high number of transcripts from frozen tissues. the reaction volumes were miniaturized with the aid of the Mosquito HV robot. Per well, 2190 nL of Lysis Buffer 2 (LB2) were dispensed. The final volume of the mixture of Lysis Buffer (LB1 and LB2) was 3.125 μL, containing NP40 2%, Triton-X100 1%, 1/300,000 diluted ERCC RNA spike-in, 3′ SMART-seq CDS Primer II A and RNAse-free water. ERCC spike-ins (Thermo Fischer Scientific, Ref. 4456740; Lot no. 00892098) were diluted 1 in 10, with RNAse-free water with 0.4 U/μL Recombinant RNase Inhibitor (Takara Clontech, Ref. 2313A) and a fresh dilution of 1 in 300,000 was prepared before the first strand synthesis. Reverse transcription and Pre-PCR amplification steps were followed as described by the manufacturer with four-times reduced volumes for all steps. Sequencing libraries were prepared using the Illumina Nextera XT DNA Sample Preparation kit (Illumina, Ref. FC-131-1096) and the combination of 384 Combinatorial Dual Indexes (Illumina- Set A to D, Ref. FC-131-2001 to FC-131-2004). In brief, 500 nL of the undiluted cDNA were transferred to a new 384 well-plate containing 1500 nL of Tagmentation Mix (TD and ATM reagents). Accordingly, all Nextera XT reagents (NT, NPM and i5/i7 indexes) were added stepwise to a final library volume of 5 μL per well. The final PCR amplification was performed through 12 cycles. Once the libraries were prepared, 500 nL from each well were pooled together into a tube (total volume of ~192 μL) to perform a final AMPure XP bead (Beckman Coulter, Ref. A63882) clean-up step. For our single nuclei libraries, two consecutive clean-ups with a ratio of sample to bead 0,9X led to library sizes between 200 and 1000 bp. Each plate, counting with a total of 384 libraries, was pooled together into one final library. A total of 4 final libraries were sequenced using an Illumina NovaSeq 6000 NGS sequencer in an SP XP flowcell, in a paired-end 150 bases length.