GSM5218672: IDH-MUT MGH208_Plate2_MscRRBS; Homo sapiens; Bisulfite-Seq
Tumor cells were blocked in 1% bovine serum albumin in Hanks buffered saline solution (BSA/HBSS). Tumors were first stained first with CD45-Vioblue direct antibody conjugate (clone REA747, Miltenyi Biotec) for 30 min at 4°C. Cells were washed with cold PBS, and then resuspended in 1 ml of BSA/HBSS containing 1 μM calcein AM (Life Technologies) and 0.33 μM TO-PRO-3 iodide (Life Technologies) to co-stain for 30 min before sorting. CD45 negative cells were stained with calcein AM (Life Technologies) for viability and CD24-APC (human antibody, clone REA832, Miltenyi Biotec) and CD44-VioBlue (human antibody, clone REA690, Miltenyi Biotec) to sort subpopulations of viable non-immune cells. Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488nm (calcein AM, 530/30 filter), 640nm (TO-PRO-3 or CD24-APC, 670/14 filter), and 405nm (CD45-VioBlue or CD44-VioBlue, 450/50 filter) lasers. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein AM positive and TO-PRO-3 negative. We sorted individual, viable, immune and non-immune single cells into 96-well plates containing TCL buffer (Qiagen) with 1% beta-mercaptoethanol. Plates were frozen on dry ice immediately after sorting and stored at -80°C prior to joint multiplexed single-cell reduced representation bisulfite sequencing (MscRRBS) and whole transcriptome library preparation and sequencing. MscRRBS and whole-transcriptome library preparation and sequencing were performed as previously described. Briefly, genomic DNA (gDNA) and mRNA have been separated manually as previously described. A modified oligo-dT primer (5′-biotin-triethyleneglycol-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, where V is either A, C or G, and N is any base; IDT) was conjugated to streptavidin-coupled magnetic beads (Dynabeads, Life Technologies) according to the manufacturer's instructions. To capture polyadenylated mRNA, we added the conjugated beads (10 μl) directly to the cell lysate and incubated them for 20 min at room temperature with mixing to prevent the beads from settling. The mRNA was then collected to the side of the well using a magnet, and the supernatant, containing the gDNA, was transferred to a fresh plate. Single-cell complementary DNA was amplified from the tubes containing the captured mRNA according to a variation of the Smart-Seq2 protocol using molecular crowding to increase sensitivity. After amplification and purification using 0.8X SPRI beads, 0.5ng cDNA was used for Nextera Tagmentation and library construction. Library quality and quantity were assessed using Agilent Bioanalyzer 2100 and Qubit, respectively. Genomic DNA present in the pooled supernatant and wash buffer from the mRNA isolation step was concentrated on 0.8X SPRI beads and eluted directly into the reaction mixtures for Msp1 (±HaeIII) (Fermentas) enzymatic reaction (10mL final reaction) for 90 min at 37°C. Heat-inactivation was performed for 10 min at 70°C. Digested DNA was filled-in and A-tailed at the 3' sticky ends in 8.5 mL final volume of 1X CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas). Reaction was supplemented with 1 mM dATP and 0.1 mM dCTP and 0.1 mM dGTP (NEB) and performed as follows in a thermocycler: 30°C for 25 min, 37°C for 25 min and heat-inactivation at 70°C for 10 min. Custom barcoded methylated adaptors (0.1 mM) were then ligated overnight at 16°C with the dA-tailed DNA fragments in the presence of 800 units of T4 DNA ligase (NEB) and 1 mM ATP (Roche) in a final volume of 11.5 mL of 1X CutSmart buffer. T4 DNA ligase heat-inactivation was performed at 70°C for 15 min the next day. Genomic DNA from 24 individual cells were pooled together according to their barcodes, giving, for a 96-well plate, 4 pools of 24 cells. Pooled genomic DNA was cleaned-up and concentrated using 1.8X SPRI beads (Agencourt AMPure XP - Beckman Coulter). Each pool was then sodium bisulfite converted (Fast Epitect Bisulfite, Qiagen) following manufacture recommendations. To ensure full bisulfite conversion, two cycles of conversion were performed. The double-stranded DNA was first denatured 10 min at 98°C and then incubated for 20 min at 60°C. 100 ng of dephosphorylated and sheared bacterial DNA was added as carrier to every pool prior to conversion. Converted DNA was then amplified using primers containing Illumina i7 and i5 index. Following Illumina pooling guidelines, a different i7 index was used for every 24-cell pool, allowing multiplexing of 96 cells for sequencing on one Illumina HiSeq lane. Library enrichment was done using KAPA HiFi Uracil+ master mix (Kapa Biosystems) and the following PCR condition was used: 98°C for 45 secs; 6 cycles of: 98°C for 20 secs, 58°C for 30 secs, 72°C for 1 min; followed by 12 cycles of: 98°C for 20 secs, 65°C for 30 secs, 72°C for 1 min. PCR was terminated by an incubation at 72°C for 5 min. Enriched libraries were cleaned-up and concentrated using 1.3X SPRI beads. DNA fragments between 200 bp and 1 Kb were size-selected and recovered after resolving on a 3% NuSieve 3:1 agarose gel. Libraries molarity concentration calculation was obtained by measuring concentration of double stranded DNA (Qubit) and quantifying the average library size (bp) using an Agilent Bioanalyzer. Every 24-cells pool was mixed with the others pool in an equimolar ratio. All cells from a 96-well plate were sequenced as paired-end on HiSeq 2500 with 10% PhiX spike-in. Negative controls (empty wells with no cells) were used to control for non-specific amplification of the libraries. Single cell bisulfite-seq (reduced representation) and single-cell RNA-seq (full-length transcriptome profiling)