title: 323mut_7838_AGTCAA_read1.fastq.gz
organism: Mycoplasmoides pneumoniae
description: Protocols: Mycoplasma pneumoniae cells were washed twice with PBS and lysated with 700 l of Qiazol buffer. RNA extractions were performed by using miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer. Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 g of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5 and 3 ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3 adapters and subsequently 5 adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3 RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries pas performed using 6% Novex TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 l of elution buffer.
attributes.tag: ENA first public
attributes.value: 2016-01-18
attributes.tag: ENA last update
attributes.value: 2015-11-26
attributes.tag: External Id
attributes.value: SAMEA3675660
attributes.tag: INSDC center alias
attributes.value: Centre for Genomic Regulation, CRG
attributes.tag: INSDC center name
attributes.value: Centre for Genomic Regulation, CRG
attributes.tag: INSDC first public
attributes.value: 2016-01-18T17:03:19Z
attributes.tag: INSDC last update
attributes.value: 2015-11-26T09:33:19Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4081:323mut_7838_AGTCAA_read1.fastq.gz
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: Overexpression ncMPN323
attributes.tag: sample name
attributes.value: E-MTAB-4081:323mut_7838_AGTCAA_read1.fastq.gz
attributes.tag: strain
attributes.value: M129