GSM4499005: 1220_RT_cDNA; Homo sapiens; RNA-Seq
Peripheral venous blood samples were collected by venipuncture from two voluntary blood donors, one male and one female. Blood samples were collected in ACD-tubes and stored at room temperature (RT) or 4ºC. In the former condition peripheral blood mononuclear cells (PBMC) were isolated at 2, 8, 24 and 48 h. For samples stored at 4ºC, PBMC were isolated at 24 and 48hrs. PBMC separation was performed using Ficoll density gradient centrifugation. For each condition, 12 ml of blood were diluted with an equal volume of pre-warmed RPMI 1640 culture medium (Lonza). The diluted blood was then carefully layered onto a Leucosep tube (Greiner Bio‐One) prefilled with 15 ml of Ficoll-Plus (GE Healthcare Biosciences AB) and centrifuged for 15 min at 800 x g and RT (without acceleration and brake). After centrifugation, PBMC were collected with a sterile Pasteur pipette into a 50 ml tube, diluted up to 10 ml with pre-warmed RPMI medium and centrifuged for 10 min, at 400 x g and RT. Following a second washing step with 5 ml of RPMI medium and a 5 min centrifugation, PBMC were resuspended in 8 aliquots of freezing media. Freezing media consisted of RPMI 1640 with 20% heat-inactivated fetal bovine serum (Sigma-Aldrich), 10% DMSO (Sigma-Aldrich) and Penicillin-Streptomycin 1:1000 (Lonza). 1 ml aliquots, with approximately 1x10e6 cells/ml, were gradually frozen using a commercial freezing box (Mr. Frosty, Nalgene) at -80°C for 24 h and then stored in a vapor–phase liquid nitrogen tank at -150ºC. Cryopreserved (-80ºC) PBMC samples were rapidly thawed in a 37ºC water bath. Each sample was transferred into a 15 ml Falcon using a 1000 ul cut tip without mixing by pipetting. Next, 1 ml of 37ºC pre-warmed media (Hibernate-A supplemented with 10% FCS; ThermoFisher) was added drop-wise with gentle swirling of the sample. After 1 min incubation, 2 ml of pre-warmed media were added and incubated for 1 min. Next, 5 ml pre-warmed media was gently added, inverted and incubated (1 min). This step was repeated once. Finally, the samples were centrifuged at 700 x g for 5 min (4ºC). The supernatant was removed and the pellets re-suspended in 100 ul of Cell staining buffer (BioLegend). Of note, we did not observe an increase of damaged/dead cells (cell viability staining) towards the later time points with an average viability of 95% (range: 88-98%) across time points. An increased fraction of debris could be observed at 24 and 48 hours, which, however, did not result in reduced data quality (e.g. proportion of excluded cells) during data analysis (Additional file 7: Table 6). CLL patient samples were obtained from freshly extracted blood, stored either at RT or 4ºC. Mononuclear cells were isolated after Ficoll density gradient centrifugation, at 2, 4, 6, 8 and 24 h after patient blood extraction. The cells were directly cryopreserved with freezing media (RPMI 1640 with 20% FBS and 10% DMSO), in the concentration of 5-10x10e6 cells/ml, according to standarized protocol. The tumor cell content of all the samples was >80%, as assassed by immunostaining of CD19, CD20, CD5 and CD45 followed by flow cytometry. All patients gave informed consent for their participation in the study according to International Cancer Genome Consortium (ICGC) guidelines. Cell hashing was performed following manufacturer's instructions (Cell hashing and Single Cell Proteogenomics Protocol Using TotalSeq™ Antibodies; BioLegend). Therefore, samples were incubated 10 min at 4ºC with Human TruStain FcX™ Fc Blocking reagent (BioLegend). Next, sample-specific TotalSeq antibodies (anti-human Hashtag 1-8, Biolegend) were added with subsequent incubation on ice for 45 min. Cells were washed once with cold 1X PBS supplemented with 0.0005% BSA (ThermoFisher) and pelleted at 700 x g for 5 minute. A single cell solution was obtained resuspending the pellet in 1X PBS (0.0005% BSA) and filtering it through a 40 µm cell strainer. The cells were counted in an automatic cell counter (Countess v.2, ThermoFisher). Single-cell ATAC sequencing: For the single-cell ATAC-seq experiments, we analyzed one PBMC and one CLL sample isolated after 0 h, 8 h and 24 h of blood storage at room temperature before cryopreservation. Frozen samples were rapidly thawed in a 37°C water bath. Each sample was transferred into a 15 ml Falcon using a 1000 µl cut tip without mixing by pipetting. Next, 1 ml of 37°C pre-warmed media (Hibernate-A supplemented with 10% FCS) was added drop-wise with gentle swirling of the sample. After 1 min of RT incubation, additional 2 ml of pre-warmed media were added. The samples were again kept at RT for 1 min, before 5 ml of pre-warmed media were gently added. This step was repeated once. Then, samples were centrifuged at 700 x g for 5 min. Supernatant was removed and the pellets resuspended in 500 µl of PBS supplemented with 0.05% BSA. Cell concentration and viability were determined with a TC20™ Automated Cell Counter. Nuclei isolation was performed following the “Nuclei Isolation for Single Cell ATAC Sequencing demonstrated protocol” (10x Genomics). Briefly, 1,000,000 cells from the CLL sample and 300,000 cells from PBMC, were transferred to a 1.5 ml microcentrifuge tube and centrifuged at 500 x g for 5 min at 4°C. The supernatant was removed without disrupting the cell pellet and 100 µl of chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) were added and pipette-mixed 10 times. Samples were then incubated on ice during 3 min. Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20 and 1% BSA) was added and pipette-mixed. Nuclei were centrifuged at 500 x g for 5 min at 4°C and the supernatant removed without disrupting the pellet. Based on the starting number of cells and assuming a 50% loss during the procedure, nuclei were resuspended into the appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics) in order to achieve a nuclei concentration of 1,540-3,850 nuclei/µl, suitable for a Target Nuclei Recovery of 5000. The resulting nuclei concentration was determined using a with a TC20 Automated Cell Counter. Single cell 3' libraries were prepared using the 10x Genomics Single Cell 3' Reagent Kit v2, together with the 10x Chromium Controller microfluidic platform according to manufacturer's instructions (10x Genomics). In summary, our hashed samples (adjusted to 1000 cells/ul) were loaded into a 10x Chip A, together with the RT mix, barcoded beads and partioning oil. After 10x Chromium Controller processing, reaction was incubated 45 min at 53ºC and 5 min at 85ºC. Next, the cDNA was purified with silane magnetic beads. The cDNA amplification was performed using the cDNA amplification primer, plus a Hashtag oligonucleotide (HTO, Supplementary Table 4), and 25 PCR cycles. In order to separate the TotalSeq™ Hashtag fraction from the amplified cDNA a second purification was performed (see TotalSeq™-A Cell Hashing and TotalSeq™-A Antibodies Protocol for Simultaneous Proteomics and Transcriptomics with 10X Single Cell 3' Reagent Kit v2 protocol) and HTO specifically amplified and indexed (Supplementary Table 3. Finally, sequencing of HTO (5%) and cDNA (95%) libraries was carried out on a HSeq2500 (Illumina). For Smart-seq2 libraries, we profiled the transcriptome of 376 PBMC. The cells originated from the same donors as in the 10x Genomics experiments, and were distributed across four 96-well plates (all conditions per plate). We discarded 60 cells that either had <75,000 or >1,000,000 total counts, <435 detected genes or a mitochondrial expression >20%. Similarly, we filtered out 6,542 genes that had an average expression across cells <1. We normalized gene counts with the Scran package, which removed the batch effect between plates. scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Gu