title: Cryptococcus neoformans var. grubii KN99 insertion of kanMX at chr11:287563 transcriptome
design: RNA is isolated from liquid culture following the protocol for TRIzol RNA Isolation Reagent (Life Technologies 15596026). Poly-A RNA is separated using an mRNA Catcher Plus Plate (Invitrogen  K1570-02). The libraries are made by chemically shattering the RNA in TURBO DNase I (Invitrogen NC9075048) buffer at 75 degrees for 10 min. To make first-strand cDNA, shattered genomic DNA is incubating with Random Primers (Invitrogen 48190-011) and dNTP mix (NEB N0447S) at 65 degrees for 5 min. The first strand is then synthesized by using SSIII kit (Invitrogen 18080-44) 5X First Strand Buffer, DTT, and SSIII enzyme; and adding RNase OUT (Invitrogen 10777-019). It is incubated as follows: 25 degrees for 5 min, 50 degrees for 50 min, 70 degrees for 15 min, and holding at 4 degrees.The second strand is made by adding 5X SS buffer (Invitrogen 10812-014), dNTP mix (NEB N0447S), E. coli DNA ligase (NEB M02056), DNA Polymerase I (Invitrogen 18010-025), RNase H (Invitrogen 18021-014) and incubating at 16 degrees for 2 hours. Then end repair is carried out using the NEB Quick Blunting Kit (E1201S), adding an A base using Klenow Fragment (3 to 5 exo, NEB M0212S) and 1mM dATP (Invitrogen 18252-015), and finally adding the Illumina adapters and indices. Once the library is made it is sequenced on a HiSeq 2500 by Illumina.