title: GSM8977430: Donor K154 - sorted splenic B cells; Homo sapiens; OTHER
library_construction_protocol: RNA was extracted from sorted B cell lysates using the RNeasy plus micro kit (QIAGEN) as per manufacturer's instructions. Reverse transcription (RT) was performed in two steps:  First, 1uL barcoded reverse primer mix (10uM per each primer), 1uL dNTP (10mM) and 14uL RNA template (up to 500ng) were mixed and heated to 65°C for 5 mins followed by an immediate transfer to ice for at least one minute. Second, mixture 2 including 4uL 5X First-Strand Buffer, 1uL DTT (0.1M), 1uL RNaseOUTTM (Thermo Scientific) and 1uL SuperScript®III (Life Technologies) was added and incubated at 50 °C for 60 min followed by 15 min inactivation at 70 °C. cDNA was cleaned-up with Agencourt AMPure XP beads and PCR amplified with V-gene multiplex primer mix (10 μM each forward primer) and 3′ universal reverse primer (10 μM) using KAPA protocol (KAPA Biosystems) and the thermal cycling conditions: 1 cycle (95 °C—5 min); 5 cycles (98 °C—5 s, 72 °C—2 min); 5 cycles (98°C—5 s, 65 °C—10 s, 72 °C—2 min); 30 cycles (98 °C—20 s, 60 °C—1 min, 72 °C—2 min) and 1 step (72 °C—7 min). Primers are provided in Supplementary Information (Table 2).
identifiers.namespace: GEO
identifiers.id: GSM8977430_r1