GSM3602861: Mock_4 [IonXpress_042]; Homo sapiens; RNA-Seq
Single cell suspensions of huNSG-A2-derived splenocytes were subjected to lysis of erythrocytes by ACK lysing buffer (Life Technologies) followed by magnetoc cell sorting of B cells using CD19 microbeads (Miltenyi Biotech). Total RNA of CD19+ cells was isolated using the RNeasy Mini Kit (QIAGEN) and traces of genomic DNA were removed by on-column DNase digestion (Qiagen). Purified RNA was stored at −20°C. RNA quality was assessed further by an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit. All RIN numbers were ≥ 9.5. RNA was quantified by NanoDrop 1000 spectrophotometry. Reverse transcription of 10 ng total RNA, amplification of targets by ultra-high multiplex PCR, and sequencing library construction was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific; #A26325) and Ion Xpress barcodes. Unamplified libraries were quantified by qPCR and equal amounts were combined for sequencing (6 µl of 100 pM total libraries). Templating of Ion Sphere Particles by emulsion PCR was conducted using the Ion 540 Kit (Thermo Fisher Scientific #A27753) and an OT2 instrument. Sequencing using an Ion 540 semiconductor chip was carried out on the Ion S5 System (Thermo Fisher Scientific). The Ion AmpliSeq Transcriptome Human Gene Expression Kit uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. 10 ng total RNA per sample were processed, and IonXpress barcodes were used for library building.
GEO Accession
GSM3602861