title: GSM2356599: WT_pB_rep2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted following commercial Trizol® Reagent [Cat No 15596-026, Life TecnologiesTM, GibcoBRL] recommendations. Its chemical quality was measured by Nanodrop 1000 Spectrophotometer [Thermo ScientificTM] and it integrity was confirmed using the Agilent 2100 Bioanalyzer. Sorted proB cells RNA sequencing (low input RNA-seq). RNA sequencing libraries were prepared following the SMARTseq2 protocol (Picelli et al., 2013) with minor modifications. Briefly, RNA was quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and 3.2-30.6 ng of RNA was used for cDNA synthesis. Reverse transcription was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN (1µM; 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′), template-switching oligonucleotides (1µM) and betaine (1M). The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kappa Biosystems), 100 nM ISPCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and 20 cycles of amplification. Following purification with Agencourt Ampure XP beads (1:1 ratio; Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitvity DNA Kit (Agilent). 200 ng of the amplified cDNA was fragmented for 10 min at 55 ºC using Nextera® XT (Illumina) and amplified for 12 cycles with indexed Nextera® PCR primers. Products were purified twice with Agencourt Ampure XP beads (0.8:1 ratio) and quantified again on a Bioanalyzer using a High Sensitvity DNA Kit. Sequencing of Nextera® libraries was carried out using fraction of a sequencing lane on a HSeq2000 (Illumina inc.) using TruSeq SBS Kit v3-HS, and between 74-80 million paired-end reads were generated for each sample. The read length was 101bp with TruSeq Dual Index of 8bp+8bp. Images analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA 1.8.