title: GSM2408141: HEK293_Fresh_Exp_1_401U_MSCB-037; Homo sapiens; RNA-Seq
library_construction_protocol: For single cell analysis, cryopreserved cells were rapidly thawed in a water bath in continuous agitation and placed into 25 ml of cold 1x HBSS. Fresh cells were trypsinized, pelleted and resuspended in 1x HBSS. Before sorting, cells from both conditions were filtered (70 µm nylon mesh) and propidium iodide staining identified dead/damaged cells.    Whole blood was collected from a healthy donor into EDTA tubes (Becton, Dickinson and Co). Separation of peripheral blood mononuclear cells (PBMC) was performed with Ficoll-Paque PREMIUM (GE Healthcare) according to manufacturer's instructions. Briefly, for density gradient separation, 4 ml of blood was added onto 3 ml of Ficoll-Paque PREMIUM. Centrifuged at 400 x g for 30 minutes at 18ºC without brake. PBMC layer was recuperated and washed twice with PBS at 400g for 15 minutes at 18ºC. PBMC were resuspended in freezing solution (10% DMSO, 90% non-inactivated FBS) and frozen with gradually decreasing temperatures (1ºC/min) to ‑80ºC (cryopreserved). After storage for one week at -80ºC, the cryopreserved sample was rapidly thawed in a water bath in continuous agitation and placed into 25 ml of cold 1x HBSS. Cells were washed once in ice cold 1x HBSS and resuspended in DMEM before sorting. To avoid batch effects, fresh PBMC were sorted in parallel with the cryopreserved material and distributed over the same sequencing pools. Therefore, blood from the same donor was isolated as described above and directly resuspended in DMEM. Dead and damaged cells were identified by propidium iodide staining.    Primary mouse colon was dissected from an athymic nu/nu mouse and placed on ice. The sample was divided and half of the colon was immediately prepared for single cell separation, while the other half was minced on ice, placed into freezing solution II (10% DMSO, 90% non-inactivated FBS) and frozen with gradually decreasing temperatures (1ºC/min) to ‑80ºC (cryopreserved). After storage for one week at -80ºC, the sample was rapidly thawed in a water bath in continuous agitation and placed into 25 ml of cold 1x HBSS. For single cell separation the fresh and conserved samples were minced on ice and enzymatically digested in 5 ml 1x HBSS and 83 µl collagenase IV (10,000 U/ml) for 10 min at 37ºC. Single cells were separated by passing the sample through a 0.9 mm needle and filtration (70 µm nylon mesh). Cells were washed once in ice cold 1x HBSS and resuspended in DMEM before sorting. Dead and damaged cells were identified by propidium iodide staining.    We generated an ovarian orthotopic tumor model, referred as Orthoxenograft® or patient derived orthotopic xenograft (PDOX). For single cell separation the fresh and conserved samples were minced on ice and enzymatically digested in 5 ml 1x HBSS and 83 µl collagenase IV (10,000 U/ml) for 15 min at 37ºC. Single cells were separated by passing the sample through a 0.9 mm needle and filtration (70 µm nylon mesh). Cells were washed once in ice cold 1x HBSS and resuspended in DMEM before sorting. In order to enrich human cells during the sorting procedure, tumor cells were stained for 1 hour at 4ºC with α-EpCam (CD326, eBioscience, 1:100). Propidium iodide staining identified dead/damaged cells. To construct single cell libraries from polyA-tailed RNA, we applied massively parallel single-cell RNA sequencing (MARS-Seq; Jaitin et al., Science 2014).  This methodology generates paired-end reads where read 1 contains the transcript sequence and read 2 the cell and UMI barcodes. Briefly, single cells were FACS-sorted into 384-well plates, containing lysis buffer and reverse-transcription (RT) primers. The RT primers contained the single cell barcodes and unique molecular identifiers (UMIs) for subsequent de-multiplexing and correction for amplification biases, respectively.    Full-length RNA sequencing libraries were prepared using the SMARTseq2 protocol (Picelli et al., Nat Methods 2013) with minor modifications. Briefly, freshly harvested or cryopreserved (1 week at -80ºC) single cells were sorted into 96-well plates containing the lysis buffer. Reverse transcription was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN, template-switching oligonucleotides and betaine. The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kappa Biosystems), ISPCR primer and 20 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitvity DNA Kit (Agilent). 200 ng of the amplified cDNA was fragmented using Nextera® XT (Illumina) and amplified with indexed Nextera® PCR primers. Products were purified twice with Agencourt Ampure XP beads and quantified again using a Bioanalyzer High Sensitvity DNA Kit.
attributes.tag: GEO Accession
attributes.value: GSM2408141