title: GSM9037564: CR-Hlg299; Homo sapiens; RNA-Seq
library_construction_protocol: The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with 1 mg/mL collagenase I (Gibco)，1 mg/mL collagenase II，60U/mL Hyaluronidase (Sigma), 10U/mL Liberase (Roche) and 0.02mg/mL DNase I (Roche) for 90 min at 37°C, with agitation . After digestion, samples were sieved through a 100µm and 40µm cell strainer, and centrifuged at 300g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Gibco) to lyse red blood cells.  After washing with DPBS containing 0.5% BSA, the cell pellets were re-suspended in DPBS containing 0.5% BSA，stained and counted. The Chromium instrument and the Single Cell 3' Reagent kit V3.1 (Dual Index) was used to prepare individually barcoded single-cell RNA-Seq libraries following the manufacturer's protocol (10X Genomics). Briefly, sample partitioning and molecular barcoding were done on the Chromium Controller (10x Genomics), where we loaded cellular suspensions together with the Single-cell 3' Gel Beads on a Single-cell 3' chip, in which gel beads in emulsion (GEM) generation took place. RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell reagent kit V3.1 (10X Genomics).
identifiers.namespace: GEO
identifiers.id: GSM9037564_r1