title: GSM8686978: CM fresh cells; Homo sapiens; RNA-Seq
library_name: GSM8686978
library_construction_protocol: The canine insulinoma cell line canINS was cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 200 ng/mL growth hormone (GH) (ProSpec). The human insulinoma cell line CM was cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. The mouse insulinoma cell line MIN6 was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 25 mM glucose, supplemented with 15% FBS, 0.05 mM beta-mercaptoethanol (Life Technologies) and 1% penicillin-streptomycin. The rat insulinoma cell line INS-1 was cultured in RPMI-1640 + Glutamax (Gibco) supplemented with 10% FBS, 1mM sodium pyruvate (Life Tech), 10mM HEPES (Life Tech), and 0.05 mM beta-mercaptoethanol. All cell lines were cultured at 37°C with 5% CO2, and cells were passaged on reaching 70–80% confluence. Cell cryopreservation Freezing medium, consisting out of 90% FCS and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to aliquots of 1 x 106 cells/mL of the canINS and CM cell lines and cells were transferred to cryogenic vials. The vials were put into a CoolCell cell freezing vial container (Corning) and stored at -80°C overnight. Vials of the frozen cell lines were then taken out of the CoolCell containers and stored at -80°C for four weeks until use. Sample preparation Cryopreserved cells were thawed in a 37 °C water bath, after which they were washed using pre-warmed 1x phosphate-buffered saline (PBS) (Gibco) supplemented with 0.04% bovine serum albumin (BSA) (Sigma-Aldrich). Single cell suspensions of both fresh and cryopreserved cells were created at a concentration of 1 x 106 cells/mL in 1x PBS + 0.04% BSA according to the Cell Preparation for Single Cell Protocols Handbook CG00053 (10x Genomics). Using trypan blue exclusion, all cell suspensions were confirmed to have a viability greater than 90%. Samples were partitioned on a Chromium Controller (10x Genomics) within 30 min of preparation. Single-cell 3' gene expression libraries were prepared for each sample using a Chromium NEXT Gem Single Cell 3 v3.1 Kit and dual index library construction kit (10x Genomics) targeting 10,000 cells per sample. Sequencing was performed on a NovaSeq 6000 sequencer (Illumina) using a 28 x 10 x 10 x 90 bp configuration.