Truseq index adapters

Total RNA was extracted from hepatic tissue using an RNAeasy extraction kit from QIAGEN®³⁰ with a DNAse cleaning step according to the manufacturer’s instructions. RNA quality was measured using Nanodrop, Qubit 2.0 fluorometer and Bioanalyzer 2100 instruments.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.

Genomic DNA was extracted from frozen bacterial pellets by phenol: chloroform extraction and RNase treatment [74 (link)], followed by quantification on a Nanodrop 2000 spectrophotometer (Thermo Scientific). PCR amplification was used to isolate Eco sgRNA sequences from mixed genomic DNA and to attach Illumina Truseq index adapters for high-throughput sequencing. Sequencing libraries were purified by gel electrophoresis on 8% TBE gels (Invitrogen Novex), stained with SYBR Gold (Invitrogen) to visualize library bands, and scalpel-excised (200-300bp region) under blue light imaging (Azure Biosystems c600). Excised libraries were gel-extracted and precipitated [75 (link)], then resuspended in nuclease-free distilled water (Invitrogen UltraPure). Library concentration was quantified on a Qubit 2.0 fluorimeter (Invitrogen) using the dsDNA high-sensitivity assay, and assayed for purity on a 2100 Bioanalyzer (Agilent) using the high-sensitivity DNA assay. Single-end sequencing was performed on an Illumina NextSeq 500 using a custom sequencing primer and a read length of 75bp. Multiplexed samples were spiked with 5% PhiX Control v3 DNA (Illumina) to account for low diversity among sgRNA sequences. See S2 Table for custom primers used for library preparation and sequencing.

Genomic DNA was extracted from frozen bacterial pellets by phenol: chloroform extraction and RNase treatment^{74 (link)}, followed by quantification on a Nanodrop 2000 spectrophotometer (Thermo Scientific). PCR amplification was used to isolate Eco sgRNA sequences from mixed genomic DNA and to attach Illumina Truseq index adapters for high-throughput sequencing. Sequencing libraries were purified by gel electrophoresis on 8% TBE gels (Invitrogen Novex), stained with SYBR Gold (Invitrogen) to visualize library bands, and scalpel-excised (200–300bp region) under blue light imaging (Azure Biosystems c600). Excised libraries were gel-extracted and precipitated^{75 (link)}, then resuspended in nuclease-free distilled water (Invitrogen UltraPure). Library concentration was quantified on a Qubit 2.0 fluorimeter (Invitrogen) using the dsDNA high-sensitivity assay, and assayed for purity on a 2100 Bioanalyzer (Agilent) using the high-sensitivity DNA assay. Single-end sequencing was performed on an Illumina NextSeq 500 using a custom sequencing primer and a read length of 75bp. Multiplexed samples were spiked with 5% PhiX Control v3 DNA (Illumina) to account for low diversity among sgRNA sequences. See Table S2 for custom primers used for library preparation and sequencing.