title: GSM4182452: H2_PR8_G_p1_BCR-seq; Mus musculus; RNA-Seq
library_construction_protocol: Germinal Center B cells (CD43 positive) from immunized mice by H1 and H2 have been collected using AutoMACS. Furthermore, the IgG1 positive-B cells were sorted with fluorescent-labeling H1 and H2 into wells of a 96-well plate as an single cell using BD FACSAria III (BD Biosciences). The single cell in lysis buffer was immediately stored at -80 degree centigrade. Single cell was dropped in 4µL/well of lysis buffer and 1µL of 10µM Oligo(dT)18 Primer. Cell lysate was incubated at 70°C for 90 sec, at 35°C for 15 sec and then put on ice for 2min. For First-Strand cDNA Synthesis, 5µL of RT mix was added to the cell lysate and incubated consecutively at 35°C for 5min, 45°C for 20min , 70°C for 10min and 4°C for chilling. For Primer Digestion, 5µL of the Primer Digestion mix was prepared, incubated at 37°C for 30 min, 80°C for 20min and 4°C for chilling. For Poly-A tailing reaction, 5µL of the Poly-A tailing reaction mix was prepared, incubated at 37°C for 50 sec, 65°C for 10min and 4°C for chilling. For 2nd strand cDNA synthesis, 25µL of the amplification mix was prepared, incubated at 95°C for 3min, 40°C for 1min, 65°C for 10min and 72°C for 5min and chilled. For cDNA amplification by Ig-specific outer primer set, 20µL of the outer amplification mix was prepared, incubated at 94°C for 2min followed by 25 cycles of 98°C for 10sec, 60°C for 30sec, and 68°C for 45sec, then chilled. For cDNA amplification by Ig-specific inner primer set, 20L of the inner amplification mix was prepared, incubated at 94°C for 2min followed by 40 cycles of 98°C for 10sec, 60°C for 30sec, and 68°C for 45sec, then chilled. Finally, 3L of the inner cDNA amplified product was used for further plate-indexing by amplification. Finally, 3µL of the inner cDNA amplified product was used for further plate-indexing by amplification. 20µL of the plate-indexing amplification mix was prepared, incubated at 94°C for 2min followed by 25 cycles of 98°C for 10sec, 60°C for 30sec, and 68°C for 45sec, then chilled. The Libraries were sequenced on the Miseq following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM4182452