GSM6602695: P13; Homo sapiens; RNA-Seq
CD40L-expressing 3T3 cells were harvested, irradiated with 30 Gy, and 1 × 104 CD40L-expressing 3T3 cells were seeded in B cell medium (RPMI 1640 (Gibco) without phenol red containing 5% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2mM L-glutamine, 50 μM β-mercaptoethanol and 20 μg/ml human apotransferrin (Sigma Aldrich; depleted for human IgG with protein G sepharose)) in 96-well flat-bottom plates (NUNC) to allow adherence overnight. The next day, 2.5 × 104 human naive (CD19+CD27-IgG-IgD+) B cells were sorted on a FACS Aria and activated using the irradiated CD40L-expressing 3T3 cells in the presence of IL-4 (100 ng/ml; Cellgenix) for six days.  After six days, activated B cells were collected and co-cultured with 1 × 104 CD40L-expressing 3T3 cells or 1 × 104 WT together with CD40L-expressing 3T3 cells (ratio 9:1) that were irradiated and seeded one day in advance (as described above), together with IL-4 (100 ng/ml) and IL-21 (50 ng/ml; Invitrogen) for five days. Sorted cells were processed using the Smart-seq2 protocol (Picelli et al., 2014) adjusted for 384-well plates. In short, 384-well plates containing single cells were thawed and cells were lysed and mRNA denatured by incubating the plates at 95°C for 3 minutes and put back on ice. Subsequently, 2.7 μl of first-strand cDNA reagent mix, containing 1x First-strand buffer, 5 mM DTT, 1 M betaine, 14 mM MgCl2, 5 U RNase inhibitor, 25 U Superscript II Reverse Transcriptase, and 1 μM Template-Switching oligonucleotides in nuclease-free water, was added to each well. Plates were incubated at 42°C for 90 minutes, 70°C for 15 minutes, and kept on hold at 4°C. Afterward, 7.5 μl of the pre-amplification mix, consisting of 1x KAPA HiFi HotStart Readymix and 0.12 μM ISPCR primers in nuclease-free water, was added to each well. Plates were then incubated as followed: 98°C for 3 minutes, 23 cycles of 98°C for 20 seconds; 67°C for 15 seconds; and 72°C for 6 minutes, then at 72°C for 5 minutes and kept on hold at 4°C. Purification of the resulting cDNA was performed using SeraMag SpeedBeads containing 19% w/v PEG 8,000 and a 1:0.8 ratio of cDNA/beads was used for cDNA precipitation. Purified cDNA was eluted in 14 μl of nuclease-free water. The quality of cDNA was randomly checked in 5% of the wells using the Tapestation High-Sensitivity D5000 assay. For the tagmentation reaction, 0.5 μl of 50-150 pg cDNA was mixed with a 1.5 μl Tagmentation mix containing 1x Tagment DNA buffer and Amplicon Tagment mix (Nextera XT DNA sample preparation kit). Plates were incubated at 55°C for 8 minutes and kept on hold at 4°C. 0.5 μl Neutralize Tagment buffer was added and incubated at room temperature for 5 minutes to inactivate Tn5. PCR amplification of adapter-ligated cDNA fragments was performed in a final volume of 5 μl containing the two index primers (Nextera XT Index kit) in a 1:5 dilution and Nextera PCR master mix. Plates were then incubated as followed: 72°C for 3 minutes, 95°C for 30 seconds, then 14-15 cycles of 95°C for 10 seconds; 55°C for 30 seconds; and 72°C for 30 seconds, then 72°C for 5 minutes and kept on hold at 4°C. 384 wells (the entire plate, including negative controls) were pooled in a single 2 ml tube (Eppendorf). Purification of cDNA was performed by adding 400 μl of cDNA in a 1:1 ratio with SeraMag SpeedBeads in 19% w/v PEG 8,000 in a 1.5 ml LoBind tube. Beads were washed with 1 ml of 80% ethanol. Purified cDNA was eluted in 200 μl of nuclease-free water. The concentration of the library was measured by Qubit according to the manufacturer's instructions. The size distribution of the library was measured using the Tapestation High-Sensitivity D1000 or D5000 assay. The cDNA library pool was stored at -20°C until ready for sequencing.   cDNA library pool was diluted to 2 nM and prepared for sequencing using the NextSeq 500 High Output Kit v2 (75 cycles) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq 500 instrument.