title: Illumina HiSeq 2000 sequencing; RNA-Seq_Mature B cell ALL_
design: design_description
library_construction_protocol: 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.
attributes.tag: LIBRARY_GENERATION_3'ADAPTER_SEQUENCE
attributes.value: NA
attributes.tag: PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL
attributes.value: Ribo-Zero Gold RNA method, with directional RNA prep for Illumina Sequencing
attributes.tag: PREPARATION_PCR_NUMBER_CYCLES
attributes.value: 0
attributes.tag: AMPLIFIED
attributes.value: TRUE
attributes.tag: LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
attributes.value: Kapa High-Fidelity DNA Polymerase (Kapa Biosystems)
attributes.tag: LIBRARY_GENERATION_FRAGMENTATION
attributes.value: 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.
attributes.tag: LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
attributes.value: PCR Purification Kit using  MinElute  column (Qiagen)
attributes.tag: LIBRARY_GENERATION_PCR_TEMPLATE_CONC
attributes.value: NA
attributes.tag: LIBRARY_GENERATION_PCR_PRIMER_CONC
attributes.value: 25µM
attributes.tag: LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
attributes.value: CAAGCAGAAGACGGCATACGAGAT
attributes.tag: LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
attributes.value: AATGATACGGCGACCACCGAGATCTACAC
attributes.tag: LIBRARY_GENERATION_PROTOCOL
attributes.value: 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)
attributes.tag: TEMPLATE_TYPE
attributes.value: cDNA
attributes.tag: EXPERIMENT_TYPE
attributes.value: mRNA-Seq
attributes.tag: LIBRARY_GENERATION_FRAGMENT_SIZE_RANGE
attributes.value: 290-350
attributes.tag: LIBRARY_GENERATION_PCR_NUMBER_CYCLES
attributes.value: 10
attributes.tag: EXTRACTION_PROTOCOL
attributes.value: RNA was extracted using Trizol reagent(Invitrogen) and purified with RNeasy Mini kit(Qiagen).
attributes.tag: LIBRARY_GENERATION_5'ADAPTER_SEQUENCE
attributes.value: NA
attributes.tag: PREPARATION_INITIAL_RNA_QNTY
attributes.value: 500ng
attributes.tag: LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
attributes.value: 30" 98ºC, [40" 98ºC, 30" 65ºC, 30" 72ºC]10 cycles,  5' 72ºC, hold 4ºC