title: RNA-Seq of Mus musculus: rostral and caudal forelimb corticospinal neurons
design: SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara #634440) was used per manufacturers instructions for cDNA synthesis of single cell RNA and subsequent amplification, with the exception that all reaction volumes were reduced to 0.5x. Single cells were stored in 8-strips at -80C in 5.25 l of collection buffer (SMART-Seq v4 lysis buffer at 0.83x, RNase Inhibitor at 0.17 U/l, and ERCC MIX1 at final 1x10-8 dilution as described above). Twelve to 36 8-strips were processed at a time (the equivalent of 1-3 96-well plates). At least 1 control strip was used per amplification set, containing 2 wells without cells (termed ERCC), 2 wells without cells or ERCC (termed NTC), and 2 wells of 10 pg of Mouse Whole Brain Total RNA (Zyagen, MR-201) and 2 wells of 10 pg Control RNA provided in the Takara kit.  Mouse nuclei were subjected to 22 or 23 PCR cycles after the reverse transcription step. SPRI bead (Sera-Mag Select beads GE Healthcare #29343057) purification was done using the Agilent Bravo NGS Option A instrument. A bead ratio of 1x was used (25 l of Sera-Mag Select beads to 25 l cDNA PCR product with 0.5 l of 10x lysis buffer added, as per Takara instructions at 0.5x volume), and purified cDNA was eluted in 17 l elution buffer provided by Takara. All samples were quantitated using PicoGreen on Molecular Dynamics M2 SpectraMax instrument. The samples were then run on the Agilent Fragment Analyzer (96) using the High Sensitivity NGS Fragment Analysis Kit (1bp-6000bp) to qualify cDNA size distribution. Purified cDNA was stored in 96-well plates at -20C until library preparation. All samples proceeded through NexteraXT DNA Library Preparation (Illumina FC131-1096) using custom 8-base Unique Design Index primers designed and manufactured by IDT (Integrated DNA Technologies). NexteraXT DNA Library prep was done at 0.2x volume on the Mantis instrument (Formulatrix). Reduction in volume was applied to input and all reagents, but otherwise the manufacturers instructions were followed. 50pg cDNA input was used in generating the libraries. An aliquot of all amplified cDNA samples was first normalized to 50 pg/ul with Nuclease-Free Water (Ambion), then this normalized sample aliquot was used as input material into the NexteraXT DNA Library Prep (for a total of 50pg). Before bead purification after PCR, 12.1ul of Nuclease-Free Water (Ambion) was added to the 10ul PCR reaction volume, bringing the total volume to 22.1ul post-PCR and before bead purification.  SPRI Sera-Mag Select bead purification was done using the Agilent Bravo NGS Option A instrument. A bead ratio of 0.9x was used (20 ul of Sera-Mag Select beads to 22.10 ul library product, as per Illumina protocol), and all samples were eluted in 22 l of Resuspension Buffer (Illumina). All samples were quantitated using PicoGreen using Molecular Bynamics M2 SpectraMax instrument. All samples were run on the Agilent Fragment Analyzer (96) using the High Sensitivity NGS Fragment Analysis Kit (1bp-6000bp) for sizing. Molarity was calculated for each sample using average size as reported by the Fragment Analyzer and pg/l concentration as determined by PicoGreen. Samples (5 l aliquot) were normalized to 2-5 nM with Nuclease-free Water (Ambion), then 2 l from each sample within one 96-index set was pooled to a total of 192 l at 2-5 nM concentration. Libraries were further multiplexed at 768 samples/flowcell by pooling 8 96-sample libraries using compatible Index Sets. A portion of the final library pool was sequenced on an Illumina NextSeq2000 instrument using the P2 flowcell, for a target of 500,000 reads per single cell or nucleus.