GSM4862202: AB7304; Mus musculus; RNA-Seq
Mice were euthanized and spinal cord meninges and cervical LNs were disected. cLN were digested in IMDM (Sigma-Aldrich) media supplemented with Liberase-TL (100μg/ml, Roche) and DNase-I (100μg/ml, Roche), and spinal cord meninges were digested in RPMI (Sigma-Aldrich) media supplemented with collagenase IV (40U/ml, Roche) and DNase-I (100μg/ml, Roche). Both tissues were incubated with frequent agitation at 37oC for 20 min. After the dissociation procedures, cells were washed with sorting buffer, filtered through 100μm, 70μm and 40μm cell strainers, and centrifuged at 380g, 5min, 4oC. Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014}