alias: SRA-532fd24b-a363-4017-899d-c10f5b2a2026
design: Total RNA was extracted from the sorted cells with RNAeasy kit (QIAGEN) according to the manufacturerÃ¢ÂÂs protocol. Amplicons for sequencing on the Illumina MiSEQ platform were synthesized using commercial reagents and a protocol provided by the manufacturer (New England Biolabs, Ipswich MA). Briefly, extracted RNA (100 ng) was reverse-transcribed into cDNA using a biotinylated oligo dT primer. An adaptor sequence was added to the 3Ã¢ÂÂ end of all cDNA, which contains a universal priming site and a 17-nucleotide unique molecular identifier (UMI). Products were purified using streptavidin-coated magnetic beads followed by a primary PCR reaction using a pool of primers targeting the IGHA, IGHD, IGHE, IGHG, IGHM, IGKC, and IGLC regions, as well as a primer against the universal adapter sequence. The Ig-specific primers contained tail sequences for the later Illumina adapter addition. PCR products were then purified using AMPure XP beads. A secondary PCR was then performed to add the Illumina P5 adapter to the C region end and a sample-indexed P7 adapter to the universal adapter end. The number of secondary PCR cycles was tailored to each sample to avoid entering the plateau phase, as judged by a prior quantitative PCR analysis. Final products were purified, then quantified with a TapeStation (Agilent Genomics).
identifiers.namespace: CEDAR
identifiers.id: SRA-532fd24b-a363-4017-899d-c10f5b2a2026
identifiers.namespace: CEDAR
identifiers.id: SRA-532fd24b-a363-4017-899d-c10f5b2a2026