GSM3780793: Im-Rep2; Mus musculus; RNA-Seq
Naive or immunized (day 35) C57BL/6 mice were injected intravenously with 2.5 ug Alexa 700 conjugated anti-CD45.2 Ab in PBS, and after 7 min, mice were euthanized. For in vitro CD45.2 staining, PECy7 conjugated anti-CD45.2 Ab was used. For isolation of total lung cells, cells were collected after 1 hr digestion in RPMI media supplemented with 5% FCS, 1 mg/ml Collagenase D (Roche), 10 mg/ml DNase I (Sigma) and 0.1 mg/ml Dispase II (Sigma) at 37 C 250 rpm. Homogenates were passed through a cell strainer, and ammonium- chloride-potassium lysing buffer was used. After in vitro staining with PECy7 anti-CD45.2 and Pacific blue anti-CD4 Abs, CD45.2 in vivo neg , CD4 neg   cells were sorted into PBS plus 0.01% BSA. The cells were diluted to concentration of 100 cells/μl and 1 ml aliquots were used as input to the Drop-seq protocol. Cells were processed for Drop-seq within ∼30 min of collection. The beads were purchased from ChemGenes Corporation, Wilmington MA (catalog number Macosko201110) and the PDMS co-flow microfluidic droplet generation device was generated by Nanoshift, Emeryville CA. The cDNA was generated using Template Switch Oligo and the populations of 5,000 beads (∼150 cells) were separately amplified for 16 cycles of PCR (conditions identical to those previously described) with SMART PCR primer and  PCR products were purified by the addition of 0.6x AMPure XP beads (Beckman Coulter). For each conditions the cells from multiple mice were pooled together. Immunized group consisted of cells from 3 mice and naïve condition consisted of 5 mice. Each sample was collected by Drop-seq. For immunized conditions two 1 ml collections were performed and for naïve mouse one collection was performed. For immunized group the cDNA from an estimated 4000 cells was prepared and tagmented by Nextera XT using 1000 pg of cDNA input, and the custom primers New-P5-SMART-hybrid [3] and Nextera XT primers - N701 and N702 (Illumina). For naïve group, cDNA from an estimated 2000 cells was used as input into the Nextera XT tagmentation with New-P5-SMART-hybrid oligoand N704. The concentration and the quality of libraries was measured with Qubit HS DNA assay and Agilent TapeStation Three libraries were sequenced on the Illumina NextSeq 500 using 2.0 pM in a volume of 1.3 ml HT1, and 2 ml of 0.3 μM Custom Read 1 primer (3) for priming of read 1. Read 1 was 20 bp; read 2 (paired end) was 60 bp.