title: GSM6721761: Bone marrow, CD, 7m, rep1; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow was flushed and kept in MACS Tissue Storage Solution (Miltenyi Biotec). Samples were sieved through 40-μm cell strainers and centrifuged at 300 g for 5 min. Pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) for lysing red blood cells. Brain tissues were surgically removed and minced into small pieces (approximately 1 mm ) on ice and digested by 200 U/ml Papine (Diamond) and Cysteine-HCL (Sigma). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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.