GSM4405549: MeV F 1st library in pCDNA3 with high mutation rate; Measles morbillivirus; OTHER
Trizol Total cytosolic RNA were reverse transcribed by F gene specific primer. Then amplicons were amplified by PCR targeting 1st quarter, 2nd quarter, 3rd quarter, and 4th quarter of F gene. Cytosolic RNAs were extracted by Trizol, then RNA were reverse transcribed by superscript III (invitrogen) with a primer set of  'forward; ACTCATCCAGTGTCCATCATG' and 'reverse; TGTTTCAAGAGTTGTAGAGGATCA' to amplify entire F gene.   Using 1st amplicon as a template, we amplified part of F gene (1st, 2nd, 3rd, 4th quarter) by using cloneamp (takara bio).   Using 1st amplicon as a template, we amplified part of F gene (1st, 2nd, 3rd, 4th quarter) by using cloneamp (takara bio).  1st-f; CCAGTGTCCATCATG, 1st-r; CTTGAGAGTTCAGCA, 2nd-f; CACTTCACCAGTCCA, 2nd-r; TAGGCTATACTGAGT,  3rd-f; GACACAGAGTCCTACTTCATTGT, 3rd-r; GCAGTGATCGGCAGCAATG, 4th-f; GACAAGATCCTAACATA, 4th-r; AAGAGTTGTAGAGGATCA.  DNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). DNA Library Preparation, clustering, and sequencing reagents were used throughout the process using NEBNext Ultra DNA Library Prep kit following the manufacturer s recommendations (Illumina, San Diego, CA, USA). End repaired adapters were ligated after adenylation of the 3 ends. The pooled DNA libraries were loaded on the Illumina instrument according to manufacturer s instructions.  The samples were sequenced by MiSeq on a 2x 250 paired-end (PE) configuration. Base calling was conducted by the Illumina Control Software (HCS) on the Illumina instrument.