title: GSM4626851: p27minusmacromet; Homo sapiens; RNA-Seq
library_construction_protocol: The brain was removed and placed in ice cold Hank's Balanced Salt Solution (HBSS) in a 10 cm dish. Fresh brain tissues were dissected under an Axiozoom fluorescent stereoscope (Zeiss) to distinguish tdTomato+ metastatic lesions from apparently uninvolved brain. The metastatic and uninvolved brain tissue were separated and minced using a razor blade, then enzymatically dissociated with papain (2 mg/ml) (Worthington Biochemical Corporation) and 0.06 mg/ml DNase I (Worthington Biochemical Corporation) in HBSS. A single cell suspension was obtained by incubating samples at 37oC for 30 minutes with agitation every 5 minutes. The cell mixture was filtered through a 70 μm cell strainer to remove unlysed tissue fragments and single cells were pelleted at 500 x g for eight minutes. The cell pellet was resuspended immediately in 1 ml fresh RBC lysis buffer (Biolegend) to remove red blood cells. Lysis was neutralized by adding 1-3 volumes of HBSS and 10% FBS, then cells were pelleted and resuspended in 10 ml 30% Percoll (GE Healthcare). The sample was centrifuged at 700 x g using a swinging bucket rotor for 10 minutes to remove myelin (breaks set to 0, acceleration set to 1). The cell pellet was resuspended in 200 μl of 2% FBS/HBSS and filtered through a 40 μm cell strainer before flow sorting. Single-cell libraries were prepared using the Chromium 3′ v2 platform (10X Genomics) following the manufacturer's protocol 7. Briefly, single cells were partitioned into gel beads in emulsion in the 10X Chromium Controller instrument followed by cell lysis and barcoded reverse transcription of RNA, amplification, shearing and 5′ adapter and sample index attachment. Cells from the three groups indicated above were loaded separately onto the Chromium Chip A for indexing before libraries were combined for paired-end sequencing on the Illumina HiSeq2500 (PE1 54 bp and PE2 66 bp).
attributes.tag: GEO Accession
attributes.value: GSM4626851