GSM4187034: Upper layer non-astrocytes Replicate 2; Mus musculus; RNA-Seq
Microdissected cortical layer tissue were minced with a forceps and enzymatically dissociated with papain (20 U/mL) in dissociation buffer (glucose 22.5 mM, EDTA 0.5 mM, phenol red), L-cysteine (1 mM) and DNase (125 U/mL) for 80 min at 33oC. Tissue was then washed in inhibitor solution (dissociation buffer with ovomucoid (1.0 mg/mL)) and centrifuged for 5 min at 200 g. Supernatant was discarded, the tissue was resuspended in the inhibitor buffer and mechanically disrupted using a P1000 pipette. Dissociated cells were layered onto inhibitor buffer with concentrated ovomucoid (5 mg/mL) and centrifuged 5 min at 200 g. Finally, the cell pellet was resuspended in staining medium with DAPI. Aldh1l1-GFP+ and Aldh1l1-GFP- cells were sorted on a BD FACS Aria II and gated on forward/side scatter, live/dead by DAPI exclusion, and GFP, using GFP and DAPI controls to set gates for each experiment. Total RNA from FACS-purified cortical layer astrocytes and whole cortical layers was extracted with Trizol LS (Invitrogen) and purified using the RNeasy Kit (Qiagen). cDNA was generated from full-length RNA using the NuGEN RNA-Seq V2 kit that employs the single primer isothermal amplification method to deplete ribosomal RNA, and sheared by Covaris to yield uniform size fragments. RNASeq libraries were generated using the NuGen Ultralow kit for adapters, barcoding, and amplification and purified using the Agencourt XP magnetic beads, quality controlled with an Agilent bioanalyzer, and quantified by qPCR. Five libraries were pooled per lane across three lanes for single end (SE50) sequencing ona. HiSeq4000.