GSM5049734: 215_216_5P_LP; Mus musculus; OTHER
Bone marrow cell suspensions were made by flushing two femurs and tibias with RF10-M (RPMI 1640 [Invitrogen] containing 2 mM l- glutamine, 10% fetal calf serum [Invitrogen], 1000 U-μg/mL peni- cillin–streptomycin [Fisher Scientific, Waltham, MA], 14mM 2-mer- captoethanol, 20 mM HEPES [GIBCO/Invitrogen, Carlsbad, CA]) using a 25-gauge needle. Single-cell suspensions from spleen were made by mincing the tissue with two needles in RF10-M and passing the material through a needleless syringe. Red blood cells were lysed in TBAC buffer (140 mM ammonium chloride, 17 mM Tris-base, pH 7.2) on ice for five minutes, and quenched with an equal volume of RF10-M. Cells were centrifuged and resuspended in 1mL of RF10-M and counted on a Model Z1 particle counter (Beckman Coulter, Fullerton, CA). For analysis for cell surface antigens, samples containing 1 × 106 cells were treated with Fc-block reagent (anti-CD16/CD32, clone 93; eBioscience RRID: AB_467132), and then incubated with antibody cocktail (BD Biosciences (San Jose, CA): anti-CD19-APC-Cy7 (1D3), eBioscience (San Diego, CA): anti-CD5-PE (53-7.3)) for thirty minutes on ice in the dark. PtC binding was detected by including 200 nm Fluorescein-DHPE/DOPC/CHOL liposomes (1:400 dilution of 0.59mg/mL stock; FormuMax Scientific) in the antibody cocktail. Cells were then washed with PBS4 (Dulbecco's phosphate buffered saline, 4% heat inactivated FBS), incubated with the desired streptavidin conjugate, washed again with PBS4, and finally suspended in PBS4 for bulk sorting. One to three splenic CD19+CD5-PtC-, CD19+CD5+PtC-, and/or CD19+CD5+PtC+ B cell populations were sorted from WT, dnRAG1, VH12, and dnRAG1-VH12 mice using a BD FACSAria flow cytometer (BD Biosciences). Samples were kept at 4 °C and immediately pelleted by centrifugation at 300 xg for 5 minutes. Supernatants were discarded and the pellets were resuspended in 500ul of cold Trizol (Ambion, Life Technologies) and RNA isolation was performed according to the Immunological Genome Project's (Immgen.org) protocol for “RNA extraction with Trizol”which is based on the maufacturer's instructions for Trizol extractions. The isolated RNA was subjected to first-strand cDNA synthesis using SuperScript III Reverse Transcriptase according to manufacturer's instructions with specific murine Ck and Cl primers (see Table). Second-strand cDNA synthesis was performed using Phusion HF Polymerase according to manufacturer's instructions with degenerate Vk and Vl primers to amplify specific Vk and Vl gene families2. The Ck and Cl primers contained a 15-mer unique molecular identifier (UMI) sequence to differentiate unique cDNAs, and a partial Illumina adaptor sequence for amplification and sequencing steps. The Vk and Vl primers also contained a partial adaptor sequence, but no UMI. The double-stranded cDNA (ds-cDNA) was purified twice using an equal volume of Ampure XP beads (Beckman Coulter) according to manufacturer instructions, and then subjected to 30 rounds of amplification using Illumina adaptor primers (both of which contained an Illumina-validated barcode to identify the genotype and sorted cell population for each sample using a two-dimensional array) and Platinum HF Taq (initial denaturation 95 °C 2 min; 30 cycles of amplification (95 °C 30s; 65 °C 2min; 68 °C 2min); and final extension (68 °C 7 min)). The prepared libraries were purified with 0.7x volume of Ampure XP beads, which was selected to remove smaller (<250bp) nucleic acid products from the amplified samples. Library concentration was measured on a Qubit fluorimeter using the Qubit dsDNA HS Assay kit (ThermoFisher Scientific). Average product size and purity was calculated and assessed with the Bioanalyzer High Sensitivity DNA Analysis kit for the 2100 Bioanalyzer Instrument (Agilent). Samples were pooled to create a 40nM library. The concentration of the pooled library was again determined using a Qubit fluorimeter and entered into the above equation with the average of all product lengths as the product size to determine concentration of the pooled library in nM. The library was then diluted to 4nM, denatured for 5 minutes with an equal volume of 0.2N NaOH [Sodium Hydroxide 1N Concentrate (Fisher Chemical) diluted in DEPC treated water (ThermoFisher Scientific)], then diluted to 20pM in thawed HT1 Buffer from the MiSeq Reagent Nano Kit v2 (500-cycles) (Illumina). Simultaneously, PhiX DNA (PhiX Control v3, Illumina) was diluted to 4nM with 200mM TrisCl (200mM Tris hydrochloride in nuclease free water, adjusted to pH 7), then denatured for 5 minutes in 0.2N NaOH and diluted to 20pM in HT1 buffer (included with the MiSeq cartridge). 240l of the 20pM library was combined with 24l of 20pM PhiX DNA (to compensate for potentially low sequence diversity due to the small number of targets) and 336L HT1 Buffer for a total loading volume of 600L at a final loading concentration of 8pM for the library with 0.8pM PhiX DNA (to make a 10% PhiX spike in), then added to the sample well on the MiSeq cartridge. Instrument instructions were followed for loading the flow cell, reagents, and cartridge onto the MiSeq for the paired-end 2 x 250bp sequencing run. The sample sheet (example included in supplement) was set up to Generate FASTQ only, using Nextera XT as the assay type to allow for Amplicon chemistry to be specified.