title: GSM3603455: CLL03_T1_Dual_RNA_c71; Homo sapiens; RNA-Seq
library_construction_protocol: Single cell experiments were performed by sorting DAPI negative cells in 96-well plates in 3 µL of 0.1X CutSmart buffer (New England Biolabs) per well using a BD Influx sorter (Becton Dickinson, Franklin Lakes, NJ). Normal B cells for sample B04, B05, and B06 were further index-sorted using a BD Influx sorter using the following sorting strategy: NBC (CD27-, IgM+, IgD+++), loMBC (CD27-, IgM+, IgD+), intMBC (CD27+, IgM+, IgD++) and hiMBC (CD27+, IgG+). The antibodies used were: FITC Mouse Anti-Human IgD (Clone IA6-2, BD Pharmingen), APC Mouse Anti-Human IgG (#562025, BD Biosciences), APC/Cy7 anti-human IgM Antibody (Clone MHM-88, BioLegend) and PE/Cy7 anti-human CD27 Antibody (clone O323, Bio Legend). Plates were then stored at -80C until further processing. The day of the experiment, cells were lysed for 2 hours at 50C in 1X CutSmart buffer supplemented with Proteinase K (0.2U, NEB) and Triton X-100 (0.3%, Sigma Aldrich) for a final volume of 5 µL. Proteinase K was heat-inactivated for 30 min at 75C. DNA was incubated with 10 units of the restriction enzyme Msp1 (Fermentas) in 6.5 µL final volume reaction during 90 min at 37C. Heat-inactivation was performed for 10 min at 70C. Digested DNA was filled-in and A-tailed at the 3' sticky ends in 8.5 µL 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: 30C for 25 min, 37C for 25 min and heat-inactivation at 70C for 10 min. Custom barcoded methylated adaptors (0.1 µM) were then ligated overnight at 16C 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 µL of 1X CutSmart buffer. T4 DNA ligase heat-inactivation was performed at 70C 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 were used to control for non-specific amplification of the libraries. Single cells are sorted by flow cytometry into 2.5 uL of RLT Plus buffer (Qiagen) supplemented with 1 U/uL of RNase Inhibitor (Lucigen). Sorted cells were immediately store at -80°C. Genomic DNA and mRNA have been separated manually as previously described. Single-cell complementary DNA was amplified from the tubes containing the captured mRNA according to the Smart2-seq protocol. 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.
attributes.tag: GEO Accession
attributes.value: GSM3603455