Illumina HiSeq 2000 sequencing; RNA-Seq_B prolymphocytic leukemia_
design_description
RNA was extracted using Trizol reagent and purified with RNeasy Mini kit(Qiagen).ribosomal rRNA was depleted from sample with RiboZero Gold Kit. RNA was fragmented in fragmentation buffer at 95°C for 1.5 min. cDNA synthesis was done with 1st/2nd strand directional cDNA synthesis. cDNA was passed through Genomic (custom) protocol using illumina PE adapter and PE primer.
LIBRARY_GENERATION_3'ADAPTER_SEQUENCE
NA
PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL
Ribo-Zero Gold RNA method, with directional RNA prep for Illumina Sequencing
PREPARATION_PCR_NUMBER_CYCLES
0
AMPLIFIED
TRUE
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Kapa High-Fidelity DNA Polymerase (Kapa Biosystems)
LIBRARY_GENERATION_FRAGMENTATION
rRNA depleted RNA was fragmented using 5xfragmentation buffer(200mM Tris Acetate, 500 mM Potassium Acetate, 150 mM Magnesium Acetate, pH8.2);buffer and NF-H20 were added to RNA to make RNA solution in 1x buffer,  incubated for 1.5 minutes at 95°C; reaction was stopped on ice for 10 minutes.
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
PCR Purification Kit using  MinElute  column (Qiagen)
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
NA
LIBRARY_GENERATION_PCR_PRIMER_CONC
25µM
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
CAAGCAGAAGACGGCATACGAGAT
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
AATGATACGGCGACCACCGAGATCTACAC
LIBRARY_GENERATION_PROTOCOL
upto 30 mg cDNA was end repaired and a 3' A overhang was created using Klenow exo (3' to 5' exo minus) ( (New England Biolabs)) for 30 minutes at 37ºC. The cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen)according to manufacturers recommendations. Nexflex adapter (BiooScientific, TX, USA) was ligated to the cDNA at room temperature for 15 minutes with T4 DNA ligase (Promega). Adapter-ligated cDNA was purified using the Minelute Reaction Cleanup Kit(Qiagen). Size selection was performed using the 2% E-Gel SizeSelect Agarose Gels and a 100 bp DNA marker according to manufacturers recommendations.  Adapter-ligated DNA molecules were enriched by PCR with reaction composition: 23 µl DNA,  2 µl PE PCR primer mix 25 ml Kapa HiFi polymerase (Ready Mix)
TEMPLATE_TYPE
cDNA
EXPERIMENT_TYPE
mRNA-Seq
LIBRARY_GENERATION_FRAGMENT_SIZE_RANGE
290-350
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
10
EXTRACTION_PROTOCOL
RNA was extracted using Trizol reagent(Invitrogen) and purified with RNeasy Mini kit(Qiagen).
LIBRARY_GENERATION_5'ADAPTER_SEQUENCE
NA
PREPARATION_INITIAL_RNA_QNTY
500ng
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
30" 98ºC, [40" 98ºC, 30" 65ºC, 30" 72ºC]10 cycles,  5' 72ºC, hold 4ºC