title: RNA-seq of Human cultured B-cell line: sub-clone RF33
design: PCR amplification of gene VH3-9; ilumina primers ligation and                 amlification; Next generation sequencing (NGS)Experimental libraries were prepared from the cDNA made from total cellular RNA by reverse transcriptase. Up to 30 cycles of PCR were employed to amplify the cDNAs using the VH3-9 leader and JH4 primers using high fidelity polymerase  (Hot start, kappa-HiFi). The JH4 primer was: 5-GGTGACCAGGGTGCCCTGGCCCCAG-3.The sequence of the JH4 primer was modified from the JH1-5 primer used in the above mentioned PCR set up in section 2.4., where the bold bases replace the multiple three (TGC) and two (TC) nucleotides in JH1-5 primer. For the vector control library, the same primers were used to amplify the plasmid control with its VH3-9 insert as above, and for the Cmu and the Gapdh control libraries the same appropriate sense and antisense primers were used as above (section 2.4.). The products were run on agarose gel to identify the single bands of the expected sizes. The double stranded cDNA products were cleaned by Agencourt AMPure XP SPRI beads (Beckman Coulter), and quantified by Qubit Fluorimeter (Thermo).Library PreparationLibraries for sequencing were prepared by end-ligation of Illumina adapters using Kappa Biosystems Library preparation kit. In Summary, Ten ng of the cleaned double-stranded cDNA, were end repaired by the addition of a single A base at each end of the molecule. Illumina adapters (Truseq) that enable attachment to the flow cell surface were then ligated to each end of the fragments. The adapters also contain unique index sequences (barcodes), which allow the libraries to be pooled, and then individually identified during downstream analysis (multiplexing). The ligated products were amplified and enriched by 8 cycles of PCR, using the specific primers for illumina Truseq adapters, according to the manufacturers protocol. Libraries were then cleaned by Agencourt AMPure XP SPRI beads (Beckman Coulter), and quantified by Qubit Fluorimeter (Thermo). Libraries were also evaluated in the TapeStation (Agilent) to check the fragment size distribution.Sequencing: The cDNA libraries were sequenced using sequencing-by-synthesis technology (Illumina).The samples were pooled together and sequenced in a Miseq flow cell, using the Illumina Miseq DNA analyzer. Pair-end sequencing was performed with a read length of 250bp X 2. We received 0.24-1.2 million raw reads for QScore Heathmap of high quality readings of 35 with ~90% of the raw reads were obtained at the 10 to ~220bp region out of the 250bp paired end length readings (R1 and R2) of each of the libraries.