GSM7068154: 5dpi_INH, bio rep 1; Mus musculus; RNA-Seq
The lesioned grey matter of the somatosensory cortex of C57BL/6J male mice (8-12weeks old)  at 3dpi and 5dpi or the corresponding region of the noninjured cortex were isolated using a biopsy punch  ( ∅ 0.25cm ,6 cortical punches from 3 animals) and the cortical cells were dissociated at a single cell level using the Papain Dissociation System (Worthington, # LK003153) followed by the Dead Cell Removal kit (Miltenyi Biotec # 130-090-101), according to manufacturer's instructions. Incubation with dissociating enzyme was performed for 60 min. Library was performed according to the manufacter's instructions (single cell 3' v2 or v3.1 protocol, 10x Genomics). Briefly, brain cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.